Compositions Comprising Human Embryonic Stem Cells and Their Derivatives, Methods of Use, and Methods of Preparation

ABSTRACT

The present invention relates to a pharmaceutical composition comprising of preparations of human embryonic stem (hES) cells and their derivatives and methods for their transplantation into the human body, wherein transplantation results in the clinical reversal of symptoms, cure, stabilization or arrest of degeneration of a wide variety of presently incurable and terminal medical conditions, diseases and disorders. The invention further relates to novel processes of preparing novel stem cell lines which are free of animal products, feeder cells, growth factors, leukaemia inhibitory factor, supplementary mineral combinations, amino acid supplements, vitamin supplements, fibroblast growth factor, membrane associated steel factor, soluble steel factor and conditioned media. This invention further relates to the isolation, culture, maintenance, expansion, differentiation, storage, and preservation of such stem cells.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This present invention relates to pharmaceutical compositions comprisinghuman embryonic stem (hES) cells or their derivatives, said stem cellsbeing free of animal products, feeder cells, growth factors, leukaemiainhibitory factor, supplementary mineral combinations, amino acidsupplements, vitamin supplements, fibroblast growth factor, membraneassociated steel factor, soluble steel factor and conditioned media, foruse in the treatment of presently incurable, terminal and medicaldiseases, conditions or disorders. More particularly, the inventionrelates to methods of treatment of clinical disorders and terminal orpresently incurable conditions using hES cells via a transplantationprotocol. The invention further relates to novel processes of preparingnovel stem cell lines which are free of animal products, feeder cells,growth factors, leukaemia inhibitory factor, supplementary mineralcombinations, amino acid supplements, vitamin supplements, fibroblastgrowth factor, membrane associated steel factor, soluble steel factorand conditioned media. This invention further relates to the isolation,culture, maintenance, expansion, differentiation, storage, andpreservation of such stem cells.

2. Background Art

A large number of human medical disorders, conditions and diseases areeither presently incurable through existing drug therapies, surgery ortransplantation methods or they are terminal.

Stem cells have the capacity to divide to generate “daughter” cells thatretain the properties of the stem cell, or to produce daughters thatbegin to differentiate into a more specialized cell type, or to produceone daughter cell of each type. Stem cells are thus central to normalhuman growth and development, and by their intrinsic characteristics arealso potential sources of new cells for the regeneration of diseased ordamaged tissue. Stem cells are present at all stages of development, andin many, (possibly most) tissues of the adult. Stem cells from differenttissues, and from different stages of development, vary in terms of thenumber and types of cells to which they normally give rise. The majorclasses of stem cells according to this classification are embryonicstem cells, somatic stem cells and embryonic germ cells.

At the earliest stages after fertilization (up to the eight cell stage),all cells of the embryo are totipotent (i.e., they have the capacity todevelop into every type of cell needed for full development, includingextra-embryonic tissues such as the placenta and the umbilical cord).After about two to five days the blastocyst stage is reached. Withinthis ball of 50-100 cells lies the inner cell mass, which will developinto the embryo proper. The inner cell mass comprises about a quarter ofall cells at this stage of development and a unique class of stem cells;the embryonic stem cells. Embryonic stem cells have the innate capacityor potential to differentiate into each of the 200 or so cell types ofthe body and are described as pluripotent. The capacity of hES cells tocontribute to all tissue types in development has not yet been fullyestablished, but they can be grown over long periods of time in cultureand expanded in number without changing their cellular genotypes orphenotypes, and maintain their pluripotent state under these conditions.

Beyond the blastocyst stage, stem cells comprise a decreasing proportionof cells in the embryo, fetus and adult body. Many if not most tissuesin the fetus and adult contain stem cells, which, in their normallocation, have the potential to differentiate into a limited number ofspecific cell types in order to regenerate the tissue in which theynormally reside. These stem cells (somatic stem cells) are multipotentand may have a more restricted potential than embryonic stem cells inthat they normally give rise to some but not all of the cell types ofthe human body. The main sources of somatic stem cells are the fetus andadult bone marrow and cord blood.

Embryonic stem cells serve as an excellent in vitro system for studyingcellular differentiation events, drug screening, and as a primary sourceof specialized differentiated cells for future regenerative therapeuticapplications.

The embryonic stem cells, being pluripotent, have the developmentalpotential to give rise to any differentiated cell type. Thus, a diseasethat results from the failure or deregulation, either genetic oracquired, of specific cell types is potentially treatable bytransplantation of hES cells or their derivatives by replacement of thedefective cells and regeneration of the affected organ or tissue andalso by stimulating the dormant and the dying tissue.

The transplantation of hES cells or their derivatives into the humanbody has been suggested to have the potential as a means for addressingunmet medical needs.

It has widely been considered that the transplantation of hES cells willrevolutionize the treatment of a wide variety of diseases, conditionsand disorders but, to date, studies have been restricted to preclinicalstudies in mice and primates. It is questionable whether the resultsobserved in animal models are truly representative of the events thatwould occur upon transplantation of such cells into the human.Furthermore, without clinical usage of the concept, the pharmaceuticalcompositions, protocols, routes of administration and dosages foradministration of the stem cells remain undefined and untested.Furthermore, although human stem cells derived from sources other thanan embryo or xenotransplantation of cells, tissues and organs from otherspecies have been used in the clinic; these attempts have been largelyunsuccessful or present a variety of debilitating side effects.

The present invention provides for the transplantation of apharmaceutical composition comprising hES cells and/or their derivativesinto humans suffering from a variety of presently incurable or terminalconditions, diseases or disorders.

U.S. Pat. No. 5,453,457 discloses a composition comprising non-murinemammalian pluripotent cells derived from a primordial germ cell andincluding basic fibroblast growth factor, membrane associated steelfactor, soluble steel factor and leukaemia inhibitory factor.

U.S. Pat. No. 6,800,480 claims a composition comprising undifferentiatedhES cells proliferating on an extra-cellular matrix.

US Patent Application No. 2003/0017587 discloses in vitro expansion ofundifferentiated embryonic stem cells obtained from an aborted fetus orfresh or frozen cleavage stage blastocysts using a culture medium thatdoes not require feeder cells. Once isolated, the embryonic stem cellsare introduced into a cell culture medium supplemented with growthfactors, fetal bovine serum, neuronal growth factor, leukaemiainhibitory factor, fibroblast growth factor, membrane associated steelfactor, soluble steel factor or conditioned media which are notdesirable in view of potential side effects upon transplantation into apatient. Also, the said patent application is silent on the protocol tobe used for the treatment of genetic or clinical disorders except thatthe patient requires one dose of each type of cell.

US Patent Application No. 2004/0071665 provides for a therapeutic methodemploying mammalian stem cells for treatment of cardiopathology. Theexample provides for embryonic stem cells differentiated to form acardiomyogenic cluster, cultured on feeder cells and then injected atthree sites of the heart of a mouse having myocardial infarction.

US Patent Application No. 2004/0107453 discloses a method for obtaining,maintaining and differentiating adult stem cells and their use intherapeutic treatment.

US Patent Application No. 2005/0124003 discloses a method for obtaining,maintaining and differentiating fetal stem cells and their use intherapeutic treatment.

In particular, and of particular relevance to the present invention,transplantation of embryonic stem cells in mouse models of spinal cordinjury (SCI) have clearly demonstrated their future potential as a firstline of treatment of acute SCI (McDonald et al., (1999) Nature Med.5:1410; Kerr et al., (2003) J. Neurosci. 23:5131; Roy et al., (2004)Nature Biotechnology, 22:297; Hori et al., (2003) Stem Cells, 21:405;Harper, (2004) Proc. Natl. Acad. Sci. 101:7123). Despite demonstratedefficacy in animal models, skepticism regarding graft versus hostrejection problems, the potential need for lifetime administration ofimmunosuppressors and tumour and teratoma formation have delayedauthorizations to reproduce preclinical safety and efficacy inexperimental human trials. A further problem facing the design ofclinical studies and of particular relevance to the present invention isthat, as yet, there are no established protocols or schedules ofadministration, there are no studies of what would be a therapeuticallyeffective dose, or active pharmaceutical composition, or what cell typesor cell combinations should be used.

It is therefore an object of the present invention to developpharmaceutical compositions which comprise hES cells and theirderivatives which are free of animal products, feeder cells, growthfactors, leukaemia inhibitory factor, supplementary mineralcombinations, amino acid supplements, vitamin supplements, fibroblastgrowth factor, membrane associated steel factor, soluble steel factorand conditioned media suspended in a biocompatible solution, carrier ormatrix, thus suitable for human use.

Yet another object of the present invention is to develop a protocol forthe treatment of presently incurable or terminal disorders.

Still a further object of the present invention is to develop a protocolfor the treatment of SCI (Spinal Cord Injury).

The compositions of the present invention are simple to prepare, safe,cost effective, efficient, easily transportable, scalable, have a goodshelf life, and are free from side effects such as antibody-antigenreactions, aberrant innervations, tumorigenicity, teratoma formation orgraft host rejection. Also, the present invention requires only oneembryo and hence the continuous supply of human embryos is not required.Also, the protocol for treatment according to the present invention doesnot require the use of immunosuppressors, and is not dependent upon HLAtyping, is not dependent upon race, gender or age of the treated subjectfor the effective treatment of the diseases, conditions or disorders, iswithout regression and is without the need for prior training in the artof administration. Treatment of subjects with the pharmaceuticalcompositions according to the practice of the present invention istherefore possible at any suitably equipped clinical facility worldwide.

In order to transplant human embryonic stem cells into humans fortherapeutic purposes it is important that such cells are free fromcontamination such as bacteria, viruses, prions or viroids. The adoptionof standard operational laboratory practices such as good manufacturingand good clinical practices reduce the risk of such contaminations to anacceptable level.

Risks to the patient exist, however, through existing cell culturemethodologies.

A major risk is that components of the cell culture medium, retained inthe pharmaceutical product for administration to human subjects areadministered and therefore represent a risk to the patient through asyet unanticipated side effects that could not have been anticipatedthrough “safety” studies in animal testing.

Elimination of such risk is therefore desirable.

The characteristics of an embryonic stem cell culture source foradministration to human subjects has been identified as having thefollowing design: it is capable of proliferation for an extended periodof time without differentiation, maintains a karyotype in which all ofthe characteristics of the donor are retained faithfully during culture,maintains the potential to differentiate into derivatives of theendoderm, mesoderm and ectoderm throughout the culture, will notdifferentiate when cultured in the absence of exogenous factors, willnot give rise to teratomas, will not be immunogenic, will not formaberrant connections and ectopic tissue, will act on the damaged tissueand not divide continuously in vivo but as they are programmed to do ina natural life cycle. There should be no contaminant present in theculture methodology and the cell line disclosed in the invention.

The major thrust of research until now has been to develop cultureconditions that meet these requirements, but to date, no such conditionshave been forthcoming or validated through clinical trials. Inparticular, the research to date has been focused on elimination of therequirement for mouse feeder cells as a matrix for the growth andde-differentiation of a human embryonic stem cell culture. The partialremedy of providing unknown growth factors through the removal of feedercells and the supplementation of “conditioned media” has also revealedan unacceptable risk in the ideal culture medium. Human embryonic stemcells cultured in the presence of feeder conditioned media still retainan inherent risk of contamination and therefore an unacceptable riskduring transplantation into humans.

An additional factor in the design of culture conditions of humanembryonic stem cells destined for administration to humans is thequestion of residual exogenous supplements in the media which may bepresent in the pharmaceutical composition administered but that areessential during the phase of cellular expansion.

These include basic fibroblast growth factor, leukemia inhibitoryfactor, membrane associated steel factor, soluble steel factor, serum,albumins or albumin supplements, amino acid supplements, vitaminsupplements, transferrins or transferrin supplements, antioxidants,insulin or insulin substitutes, collagen precursors or collagenprecursor substitutes, trace elements, residues of “conditioned media”,animal products, feeder cells, growth factors, supplementary mineralcombinations, amino acid supplements, and vitamin supplements.

Residues of such additional supplements are viewed as unnecessary risksto the safety of patients during the transplantation of human embryonicstem cells into such subjects. In addition, the supplementation of suchfactors into the culture medium adds to the risk of contamination fromthe environment and adds to the future cost of stem cell therapy andtherefore limits its applicability in a wide range of medical diseases,conditions or disorders.

A number of approaches have been adopted to reduce these risksincluding: U.S. Pat. and Application Nos. 5,843,780; 5,690,926;6,642,048; 6,800,480; 5,166,065; 6,200,806; 5,453,357; 6,090,622;6,562,619; 6,921,632, 2006/0073587 and 2002/076747.

However, none of these approaches offer a system for the production of apharmaceutical product containing human embryonic stem cells and theirderivatives which is free of potentially contaminating factors thatcould affect the efficacy and safety of human embryonic stem cells andtheir derivatives upon administration to humans.

It is therefore another objective of the invention to develop asimplified cell culture system for the expansion of hES cells and theirderivatives in a substantially undifferentiated state in order toproduce a pharmaceutical product that is ready to use in a wide varietyof medical disorders.

More particularly, it is an objective of the invention to provide aculture technique which produces a stem cell line free from animalproducts, feeder cells, growth factors, leukaemia inhibitory factor,supplementary mineral combinations, amino acid supplements, vitaminsupplements, fibroblast growth factor, membrane associated steel factor,soluble steel factor and conditioned media.

SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions comprisinghES cells and/or their derivatives, and for the use of hES cells andtheir derivatives in treating a wide variety of conditions, diseases anddisorders wherein the stem cells are introduced into the human body by avariety of routes of administration, topical applications orintralesional insertion.

The present invention further provides a method for treating a subjectsuffering from a terminal or presently incurable disease, disorder orcondition comprising a schedule of administration of a therapeuticallyeffective amount of hES cells or their derivatives via intramuscular,intravenous, caudal, intravitreous, intrastriatal, intraparenchymal,intrathecal, epidural, retrobulbar, subcutaneous, intracardiac,intracystic, intra-articular or intrathecal injection, epidural catheterinfusion, sub arachnoid block catheter infusion, intravenous infusion,via nebulizer, via spray, via intravaginal routes, via local eye and eardrops, and a schedule for administration of the hES cells and theirderivatives topically or intralesionally.

It is preferable to use hES cells or their derivatives which are free ofanimal products, feeder cells, growth factors, leukaemia inhibitoryfactor, supplementary mineral combinations, vitamin supplements, aminoacid supplements, fibroblast growth factor, membrane associated steelfactor, soluble steel factor and conditioned media, to avoid any chancesof contamination and possibilities of negative side-effects. The hEScells and their derivatives can be obtained through any known andapproved cell culture methodology, which is feeder cell free, and freefrom contamination from any source and safe for human transplantation.hES derivatives include further differentiated cells from the humanbody.

The present invention further provides pharmaceutical compositions forthe treatment of terminal or presently incurable diseases, disorders, orconditions comprising a therapeutically effective amount of hES cellsand/or their derivatives, wherein said hES cells or their derivativesare free of animal products, feeder cells, growth factors, leukaemiainhibitory factor, supplementary mineral combinations, amino acidsupplements, vitamin supplements, fibroblast growth factor, membraneassociated steel factor, soluble steel factor and conditioned media,suspended in a pharmaceutically acceptable biocompatible solution or anyother carrier vehicle.

The present invention also includes hES cells and/or their derivativesfree of animal products, feeder cells, growth factors, leukaemiainhibitory factor, supplementary mineral combinations, amino acidsupplements, vitamin supplements, fibroblast growth factor, membraneassociated steel factor, soluble steel factor and conditioned media,entrapped in a biocompatible material or matrix. The biocompatiblematerial or matrix may be selected from biopolymers, includingpolypeptides or proteins, polysaccharides, including fibronectin,various types of collagen, laminin, keratin, fibrin, fibrinogen,hyaluronic acid, heparin sulfate, chondroitin sulfate, agarose orgelatin.

The compositions of the present invention may be in a ready-to-use drugform in which the stem cells have adequate viability, i.e., they have aviability high enough to be useful in one or more methods of the presentinvention. In one embodiment, the stem cells have a viability of greaterthan about 40%, e.g., greater than about 50%, 60%, 70%, or 80%. Thecompositions may further include an antimicrobial agent, antibacterialagent, hormonal product or other pharmaceutical agent.

In order to prepare the compositions, about 750,000 to about 160 millionhES cells and/or one or more of their derivatives such as hematopoieticstem cell progenitors, neuronal stem cell progenitors, mesenchymal stemcell progenitors, insulin producing stem cell progenitors, hepatocytestem cell progenitors, cardiac stem cell progenitors, epithelial stemcell progenitors or mixtures thereof are suspended in about 0.25 ml toabout 100 ml of a carrier vehicle. In one embodiment, about 750,000 toabout 80 million hES cells are suspended in about 0.25 ml to about 10 mlof the carrier vehicle. Enrichment for specific differentiated stem cellprogenitor types in a population is a priority, although a proportion ofundifferentiated stem cells will remain in the composition. In oneembodiment, the portion of undifferentiated stem cells will be no morethan about 80% of the total population of cells. In another embodiment,the portion of undifferentiated stem cells will be no more than about40% of the total population of cells.

Terminal diseases and other disorders or conditions that may be treatedor ameliorated according to the present invention include, withoutlimitation, cancer, liver and kidney disorders, nervous systemdisorders, skin disorders, autoimmune disorders, genetic disorders, eyedisorders, musculoskeletal disorders, fertility and reproductivedisorders and cardiovascular disorders and without limitation includeAcute Myeloid Leukaemia, Adenocarcinoma, Arthritis, Astrocytoma,Auditory Nerve Atrophy, Autism, Auto Immune Disorders, Alzheimer'sdisease, Ankylosing Spondylitis, Becker's Muscular Dystrophy, BrainDamage, Burns, Cerebrovascular Insult, Cerebral Palsy, Coma, CornealUlcers, Corneal Graft Rejection, Cortico-Basal Degeneration of theNervous System, Coronary Artery Disease, Diabetes, Dementia, DownsSyndrome, Duchenne's Muscular Dystrophy, End-Stage Renal Disease, Erb'sPalsy, Fascio Scapular Muscular Dystrophy, Fertility Disorders,Friedereich's Ataxia, Heart Failure, Hepatocellular Carcinoma,Hereditary Spino Motor Neuron Disease, Huntington's Chorea, Krabbe'sDisease, Limb Girdle Dystrophy, Liver Cirrhosis, Macular Degeneration,Mental Retardation, Multiple Sclerosis, Motor Neuron Disease, MyocardialInfarction, Nephrotic Syndrome, Niemann Pick Disease, Non-HealingUlceration of the Skin, Olivo-Ponto Cerebellar Atrophy, Optic NerveAtrophy, Parkinson's Disease, Post Electric Shock Encephalopathy,Post-Rabies Vaccine Encephalopathy, Pressure Sores, ProgressiveSupranuclear Palsy, Psoriasis, Pthysis Bulbi, RestrictiveCardiomyopathy, Retinitis Pigmentosa, Right Bundle Branch Block,Sarcoidosis, Sinus Bradycardia, Spinal Muscular Dystrophy, SpinoCerebellar Ataxia, Steven Johnson's Syndrome, Systemic LupusErythematosus, Thrombocytopenia, Thalassemia, Ulcerative Colitis,Vegetative State, Cystic Fibrosis, Interstitial Lung Disease,Azoospermia, Primary Ovarian Failure, Aphthous Ulcers, HormoneImbalance, Osteo-Arthritis, Horner's Syndrome and Osteogenic Imperfecta,along with Channelopathy and Hypogammaglobulinemia.

More specifically, the present invention provides a method of treatmentof SCI of a subject comprising:

-   -   a) administering about 750,000 to about 80 million hES cells        and/or their derivatives via sub-cutaneous injection;    -   b) repeating step (a) after a pre-determined period and        thereafter administering hES cells and/or their derivatives via        intramuscular injection;    -   c) administering a therapeutically effective amount of hES cells        and/or their derivatives, wherein said cells comprise neuronal        stem cell progenitors and hematopoietic stem cell progenitors,        via intravenous injection or infusion;    -   d) administering a therapeutically effective amount of hES cells        and/or their derivatives, wherein said cells comprise neuronal        stem cell progenitors, via epidural injection and repeating said        dose after a pre-determined period depending upon the condition        of the subject as assessed by clinical and/or neurological        examination;    -   e) administering a therapeutically effective amount of hES cells        and/or their derivatives, wherein said cells comprise neuronal        stem cell progenitors, via caudal injection;    -   f) administering a therapeutically effective amount of hES cells        and/or their derivatives, wherein said cells comprise neuronal        stem cell progenitors, via intrathecal injection or sub        arachnoid block catheter;    -   g) administering a therapeutically effective amount of hES cells        and/or their derivatives, wherein said cells comprise neuronal        stem cell progenitors, via epidural injection or epidural        catheter;    -   h) administering a therapeutically effective amount of hES cells        and/or their derivatives via deep spinal injection on either        side of the spine; and    -   i) administering a therapeutically effective amount of hES cells        and/or their derivatives via intravenous infusion;        wherein steps (a) and (b) are carried out first and the        remaining steps may be carried out in any order. In one        embodiment, step (f) is repeated followed by step (g), at least        once, until the subject exhibits clinical signs of recovery from        said SCI.

The invention also provides for a method for treating a subject with adisease, disorder or condition comprising administering atherapeutically effective amount of hES cells and/or their derivativescultured in media free of animal products, feeder cells, conditionedmedia, growth factors, leukaemia inhibiting factor, fibroblast growthfactor, membrane associated steel factor or soluble steel factor, viaintramuscular injection or intravenous injection or epidural injectionor epidural catheter or retrobulbar injection or subcutaneous injectionor intracardiac injection or intracystic injection or intrathecalinjection or by topical application or intralesional application. In oneembodiment, the disease, disorder or condition is a terminal orcurrently incurable disease, disorder or condition.

The invention also provides for a method for treatment of developmental,degenerative, familial and traumatic nervous system disorders andcerebrovascular attack comprising administration of about 750,000 toabout 160 million hES cells and/or their derivatives, wherein said cellscomprise neuronal stem cell progenitors and hematopoietic stem cellprogenitors alone or in combination, via intravenous injection,subcutaneous injection, intramuscular injection, intrathecal injection,epidural catheter infusion and sub arachnoid block catheter infusion.

The invention also provides for a method for treatment of skin disorderscomprising administration of about 750,000 to about 160 million hEScells and/or their derivatives, wherein said cells comprisehematopoietic stem cell progenitors, via subcutaneous or intravenousinjection.

The invention also provides for a method of treatment of bed sorescomprising administration of about 750,000 to about 160 million hEScells and/or their derivatives via local or topical application and viaintramuscular injection.

The invention also provides for a method for treatment of auto immunedisorders comprising administration of about 750,000 to about 160million hES cells and/or their derivatives, wherein said cells comprisehematopoietic stem cell progenitors, via intramuscular injection, orintravenous injection, or subcutaneous injection, or intra-articularinjection or intravenous infusion or combinations thereof.

The invention also provides for a method for treatment of geneticdisorders comprising administration of about 750,000 to about 160million hES cells and/or their derivatives, wherein said cells compriseneuronal stem cell progenitors and hematopoietic stem cell progenitorsalone or in combination, via intravenous injection, subcutaneousinjection, intramuscular injection, intrathecal injection, epiduralcatheter infusion or sub arachnoid block catheter infusion orcombinations thereof.

The invention also provides for a method for treatment of gangrenecomprising administration of about 750,000 to about 160 million hEScells and/or their derivatives via intravenous injection, intramuscularinjection, or local application at the junction of viable and deadtissue or combinations thereof.

The invention also provides for a method for treatment of conditionsassociated with ageing comprising administration of about 750,000 toabout 160 million hES cells and/or their derivatives via intravenousinjection, subcutaneous injection, intramuscular injection, or localapplication in suspension or mixed in a biocompatible carrier such asgel, ointment, matrix, paste or aerosol spray.

The invention also provides for a method for treatment of DiabetesMellitus comprising administration of about 750,000 to about 160 millionhES cells and/or their derivatives, wherein said cells comprise insulinproducing progenitor cells, via intravenous or intramuscular injectionor combinations thereof.

The invention also provides for a method for treating CardiovascularDisorders comprising administration of about 750,000 to about 160million hES cells and/or their derivatives, wherein said cells comprisehematopoietic stem cell progenitors, via intravenous injection,subcutaneous injection, intramuscular injection, intracardiac injection,angiography or direct injection during surgery.

The invention also provides for a method for treatment of Liver andKidney Disorders comprising administration of about 750,000 to about 160million hES cells and/or their derivatives, wherein said cells comprisehematopoietic stem cell progenitors, albumin producing stem cellprogenitors and bilirubin producing stem cell progenitors, viaintravenous injection, subcutaneous injection, intramuscular injection,intravenous infusion, or local injection.

The invention also provides for a method for the treatment of Fertilityand Reproductive Disorders comprising administration of about 750,000 toabout 160 million hES cells and/or their derivatives, wherein said cellscomprise hematopoietic stem cell progenitors, via local intramuscularinjection, intratesticular injection or through subcutaneous skininjection near the epididymis.

The invention also provides for a method for the treatment ofMusculoskeletal Disorders comprising administration of about 750,000 toabout 160 million hES cells and/or their derivatives, wherein said cellscomprise neuronal stem cell progenitors and hematopoietic stem cellprogenitors alone or in combination, via intravenous injection,subcutaneous injection, intramuscular injection or intravenous catheterinfusion.

The invention also provides for a method for the treatment of EyeDisorders comprising administration of about 750,000 to about 160million hES cells and/or their derivatives, wherein said cells compriseneuronal stem cell progenitors, hematopoietic stem cell progenitors andmesenchymal stem cell progenitors alone or in combination, via localintravenous injection, subcutaneous injection, intramuscular injection,retrobulbar injection, intravitreous injection or topical application.In one embodiment, about 750,000 to about 160 million hES cells and/ortheir derivatives comprising neuronal stem cell progenitors areadministered via retrobulbar injection. In another embodiment, about750,000 to about 160 million hES cells and/or their derivativescomprising neuronal stem cell progenitors are administered viaintravitreous injection. In another embodiment, about 750,000 to about160 million hES cells and/or their derivatives comprising mesenchymalstem cell progenitors are applied to contact lenses for placement on theeye for the treatment of corneal abrasion (e.g., the contact lens iscultured with the hES cells and/or their derivatives to coat the contactlens with cells and the lens is then placed on the eye for about 24hours).

The invention also provides a method for treatment of Lung Disorderscomprising the administration of 750,000 to 160 million hES cells and/ortheir derivatives wherein said cells comprise hematopoietic stem cellprogenitors alone or in combination with neuronal stem cell progenitorsvia intramuscular injection, intravenous injection, spray, or nebulizer.In one embodiment 750,000 to 160 million hematopoietic stem cellprogenitors are administered via nebulizer in which 2 ml of normalsaline is added. In another embodiment 750,000 to 160 millionhematopoietic stem cell progenitors are administered via spray (puff).

The invention also provides a method for the treatment of HormoneDisorders comprising the administration of 750,000 to 160 million hEScells and/or their derivatives wherein said cells comprise hematopoieticand neuronal stem cells via intramuscular and intravenous routes.

The invention also provides a method for the treatment of AphthousUlcers and other ulcers comprising the administration of 750,000 to 160million hES cells and/or their derivatives wherein said cells comprisehematopoietic and neuronal stem cells in combination or alone viaintramuscular and intravenous routes.

The invention also provides a method for the treatment ofOsteo-arthritis of the knee and hip joint comprising the administrationof 750,000 to 160 million hES cells and/or their derivatives viaintramuscular, intravenous or intra-articular injection. In oneembodiment 750,000 to 160 million hematopoietic stem cell progenitorsare administered intra-articularly.

The present invention provides a cell culture methodology that isapplicable to the culture of hES cells and their derivatives that can beused for transplantation in humans without any side effects such asteratoma formation, tumor formation, antigenicity problems orgraft-versus-host rejection.

The design of the culture methodology is specifically for the productionof hES cells and their derivatives of sufficient viability and ofappropriate characteristics for therapeutically optimal activity aftertransplantation in humans.

In a preferred embodiment, the cell culture methodology is used for thegrowth, expansion, differentiation and storage of hES cells. Inaddition, the invention relates to ready to inject compositionscomprising hES cells and/or their derivatives that are stored inconditions of storage that are suitable for direct transplantation onthawing.

In contrast to existing cell culture methodologies for the maintenanceof hES cells in a substantially undifferentiated state, the culturemedium does not contain supplements such as basic fibroblast growthfactor, leukemia inhibitory factor, membrane associated steel factor,soluble steel factor, serum, albumins or albumin supplements, amino acidsupplements, vitamin supplements, transferrins or transferrinsupplements, antioxidants, insulin or insulin substitutes, collagenprecursors or collagen precursor substitutes, trace elements, residuesof “conditioned media”, animal products, feeder cells, growth factors,supplementary mineral combinations, amino acid supplements, and vitaminsupplements.

In further contrast to other cell culture methodologies for theexpansion of hES cells, the practice of the present invention does notlead to the formation of embryoid bodies, thereby avoiding the need forsurgical dissection. It is worth noting at this point that the productof the invention being a totally human product, xenotransplantation(e.g., animal testing) may not be necessary. The instance of the firstin vitro fertilization (IVF) gestation is a corollary example.

Unlike other cell culture methodologies, the cell culture techniques ofthe present invention allow for nearly unlimited expansion of hES cellswithout significant differentiation. A major advantage of the presentinvention is that a single embryo is sufficient to providetherapeutically effective amounts of hES cells and/or their derivativesto treat multitudes of patients. Thus, there is no need for repeatedprocurement of human embryos, and many of the ethical questionsassociated with the use of human embryos may be avoided.

A further advantage of the methods of the present invention is that thehES cells and their derivatives are universally acceptable products inimmunological terms, so no cross-matching of patients and cells isrequired and no immunosuppression is needed.

One aspect of the invention relates to methods for isolating hES cells,comprising:

(a) collecting a 2 to 7 day old embryo in minimal essential medium,(b) isolating hES cells from the embryo by mechanical means.

One aspect of the invention relates to a method of expanding humanembryonic stem cells free of animal products, feeder cells, growthfactors, leukaemia inhibitory factor, supplementary mineralcombinations, amino acid supplements, vitamin supplements, fibroblastgrowth factor, membrane associated steel factor, soluble steel factorand conditioned media, comprising the steps of:

(a) introducing human embryonic stem cells in a cell medium consistingof minimal essential medium, a progestin and a β-human chorionicgonadotropin (βhCG) agonist; and(b) incubating the stem cells at a temperature of about 34° C. to about38° C. in an environment of about 3.5% to about 6% carbon dioxide forabout 12 hours to about 48 hours.

Another aspect of the invention relates to methods of growing hES cellssuch that they are at a stage prior to the partially differentiatedstage and are free of animal products, feeder cells, growth factors,leukaemia inhibitory factor, supplementary mineral combinations, aminoacid supplements, vitamin supplements, fibroblast growth factor,membrane associated steel factor, soluble steel factor and conditionedmedia, comprising the steps of:

-   (a) introducing hES cells in a cell culture medium consisting of    minimal essential medium; and-   (b) incubating the stem cells at a temperature of about 34° C. to    about 38° C. in an environment of about 3.5% to about 6% carbon    dioxide for about 12 hours to about 48 hours.

Another aspect of the invention relates to a process of preparing aready to use human embryonic stem cell preparation for humantransplantation comprising:

(a) obtaining human embryonic stem cells free of animal products, feedercells, growth factors, leukaemia inhibitory factor, supplementarymineral combinations, amino acid supplements, vitamin supplements,fibroblast growth factor, membrane associated steel factor, solublesteel factor and conditioned media,(b) centrifuging said stem cells to obtain a pellet, and(c) suspending the pellet in a biocompatible solution.

Another aspect of the invention relates to a method of storing a humanembryonic stem cell in a viable condition comprising

(a) taking stem cells prepared by the methods of the present invention,(b) adding a cryopreservation agent, and(c) freezing the cells at −4 to −80° C.

The purpose of growing hES cells according to the practice of thepresent invention in the absence of such supplements is to reduce therisk of introducing bacterial, fungal, viral or other contaminationsinto the culture. This reduces the risk of infection and variability inthe characteristics of the cells.

A further purpose of the present invention is to provide simple, costeffective, scaleable methods for the production and expansion of hEScells and their derivatives in a form that is free from anycontaminating residues and is safe for use in humans.

The purpose of this invention is therefore to provide pharmaceuticalproducts in which the risk of administration of all co-contamination isreduced, and in which residual contamination of supplementary factors iseliminated.

A further purpose of the present invention is to provide a cell culturemethodology that reduces the risk of chromosomal aberrations or geneticinstability.

A further purpose of the present invention is to provide a cell culturemethodology that reduces the risk of teratoma formation in patients inwhich said culture is transplanted.

A further purpose of the present invention is to provide a cell culturemethodology that reduces the risk of tumour formation in patients inwhich said culture is transplanted.

A further purpose of the present invention is to provide a cell culturemethodology that reduces the risk of antigenicity problems or graftversus host rejection problems in patients in which said culture istransplanted.

The present invention specifically provides compositions comprising hEScells and their derivatives and a biocompatible medium that are in aform that is in compliance with international regulatory standards ofsafety and quality, and therefore in a form that is ready for injectioninto human patients for therapeutic purposes.

The present invention also provides a product of manufacture comprisinga suspension of hES cells and/or their derivatives suspended in abiocompatible solution and contained in a container for storage,transport or transplantation directly into a human.

The present invention also provides a method for the expansion of hEScells and their derivatives.

The present invention also provides a method for the expansion of hEScells and their derivatives and inhibition of their differentiation.

The present invention also provides a method for the growth of hES cellsand their derivatives that renders them therapeutically effective upontransplantation into a human that is suffering from a disease, conditionor disorder.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description and examples that follow, a number of terms are usedherein. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided.

It may be noted that the terms used to generally describe the presentinvention are used in a manner in keeping with their common meaning asunderstood by one of ordinary skill in the art.

“Embryonic stem cell” refers to pluripotent cells of humans (i.e., hEScells). In one embodiment, the hES cells are isolated from apre-blastocyst stage embryo. In another embodiment, the hES cells areprepared by dedifferentiation of at least partially differentiated cells(e.g., multipotent cells) and are totipotent in practice. Methods ofpreparing hES cells are well known and taught, for example, in U.S. Pat.Nos. 5,843,780, 6,200,806, 7,029,913, 5,453,357, 5,690,926, 6,642,048,6,800,480, 5,166,065, 6,090,622, 6,562,619, 6,921,632, and 5,914,268,U.S. Published Application No. 2005/0176707 and InternationalApplication No. WO2001085917.

“Fetal stem cells” refers to the stem cells derived from the fetaltissue, i.e., tissues of a developing human after implantation of theembryo in the uterus.

“Multipotent” refers to stem cells that can produce only cells of aclosely related family of cells.

“Pluripotent” refers to stem cells that are the descendants oftotipotent cells and can grow into any cell type except for totipotentstem cells.

“Totipotent” refers to stem cells that are produced from the fusion ofan egg and sperm cell. Cells produced by the first few divisions of thefertilized egg cell are also totipotent. These cells can grow into anytype of cell without exception.

“Therapeutically effective amount” is used in the specification todescribe concentrations or amounts of components such as embryonic stemcells, neuroprogenitor cells, neuronal cells, hematopoietic stem cellprogenitors, cardiac stem cell progenitors, or any other derivative ofstem cells, or other agents and mixtures thereof which are effective forproducing an intended result within the context of practicing one ormore aspects of the present invention.

The terms “transplanting” and “transplantation” are used throughout thespecification synonymously to describe the process by which embryonicstem cells and/or their derivatives according to the present inventionare delivered to the site within the human body where the cells areintended to exhibit a favorable effect in connection with the treatmentor amelioration of a disease, disorder or condition described hereinincluding, without limitation, for repairing damage to a subject'scentral nervous system, treating a neurodegenerative disease or treatingthe effects of nerve damage caused by stroke, cardiovascular disease, aheart attack or physical injury or trauma or genetic damage orenvironmental insult to the brain and/or spinal cord or other organs,caused by, for example, an accident or other activity, liver damage,autoimmune disorder, sexual or reproductive dysfunction, degeneration ofvarious parts of the eye, kidney damage, etc.

“Biocompatible solution” is used in the specification to refer tosolutions in which the hES cells and/or their derivatives are suspendedfor use in the protocol for transplantation or for any other subsequentuses. Such biocompatible solutions include saline and may furthercomprise other ingredients such as preservatives, antimicrobials, andthe like and other pharmaceutical agents.

“Biocompatible carrier” is used in the specification to refer to solidcarriers, solutions and mixtures in which the hES cells and/or theirderivatives are suspended for use in the protocol for topical treatment,transplantation or for any other subsequent uses. Such biocompatiblecarriers include gels, ointments, pastes, and aerosol sprays.

“Biocompatible container” is used in the specification to refer tocontainers (e.g., cell culture or storage containers) in which the hEScells and/or their derivatives are placed and which do not prevent thecells from being used in transplantation, i.e., do not contaminate thecells with compounds which cannot be administered to subjects. Examplesof biocompatible container materials include without limitation glass,stainless steel, and polystyrene.

A “progestin” is any natural or synthetic hormone havingprogesterone-like activity, i.e., having at least 25% of the activity ofprogesterone in one of any known assay for biological activity. Examplesinclude without limitation progesterone, dydrogesterone,medroxyprogesterone, norethisterone, levonorgestrel, norgesterel,gestodene, and drospirenone. Examples of other progestins are disclosedin U.S. Pat. Nos. 7,091,234, 7,084,151, 7,081,457, 7,071,205, 6,562,857,6,319,911, 6,245,757, 6,043,235, and 6,028,064.

“β-human chorionic gonadotrophin (βhCG) agonists” are defined as anynaturally occurring or synthetic βhCG or fragment or derivative thereofhaving at least 25% of the activity of natural βhCG in one of any knownassay for biological activity. Examples of βhCG agonists include withoutlimitation βhCG and those disclosed in U.S. Pat. Nos. 6,635,445,6,585,982, 6,583,109, 6,469,139, and 5,997,871.

“Minimal essential medium” is used in the specification to refer to acell culture medium comprising amino acids, salts, glucose and vitamins.Examples include RPMI, DMEM, EMEM, and GMEM.

“Nebulization” means the administration of a drug or substance into thelungs via the nebulizer.

Intra-articular means administration into the intra-articular space.

Retrobulbar means administration into the retrobulbar space.

Intravenous infusion means administration into the vein by using an ivfluid comprising the product or drug.

“Epidural” injection or catheter infusion means administration into theregion outside the dura mater of the meninges.

“Intrathecal” injection or catheter infusion means administration intothe innermost layer of the meninges, that is, the arachnoid matter intothe cerebro spinal fluid, which is in continuum with the brain.

“Caudal” injection means administration through the sacral membrane,which is approximately three centimeters above the tip of the coccyxwhich is in continuum with the epidural space.

“Deep Spinal Injection” means injection into the erector spinal muscleson either side of the spine.

“Intramuscular” injection means administration in between the musclesheets.

“Intravenous” injection means administration inside a vein.

“Acute SCI” means up to three months after the date of the SCI.

“Sub Acute SCI” means from three months after the date of SCI to up tonine months after the date of the injury.

“Chronic SCI” more than nine months after the date of the SCI.

“Derivatives” of hES cells include multipotent stem cells, pluripotentstem cells, adult stem cells and tissue specific stem cells and do notinclude fully differentiated cells. Examples of tissue specific stemcells may be found in the following table.

Cell Type Patent Number Adipose-derived stem cell 6,777,231 Breastepithelial stem cells 5,814,511 Endothelial stem cells 6,852,533 Dorsalroot ganglion progenitor cells 6,835,567 Hematopoietic progenitor cells6,537,807 CD34⁻, CD7⁺, Lin⁻, Lin⁻, CD45RA⁺, Hematopoietic stem cells5,061,620 Thy-1⁺ Hematopoietic stem cells 5,750,397 Thy-1⁺, CD34⁺Hematopoietic stem cells 5,840,580 CD34⁺, CD38⁻, HLA-DR⁺ Hematopoieticlymphoid and dendritic 5,972,627 cells CD34⁺, CD45RA⁺, CD10⁺;Hematopoietic dendritic cells CD34⁺, CD45RA⁺, CD10⁺, Hematopoietic stemcells 5,876,956 c-kit⁻, Thy-1⁻ Hematopoietic stem cells 5,681,559 CD34⁺Hematopoietic stem cells 5,677,136 HCC-1⁺ Hematopoietic progenitor cells5,858,782 CD34⁺, galactose-specific lectin⁺ Hematopoietic stem cells(quiescent) 5,807,686 CD34⁺ Keratinocyte stem cells 6,485,971 Liver stemcells 6,129,911 Do not express OC2 6,872,389 Lymphohematopoieticprogenitor stem 5,256,560 cells My10⁻ Lymphoid progenitor cells6,908,763 Mesencephalon neural progenitor cells 6,913,925 Mesenchymalstem cells 6,387,367 CD45⁺ Mesenchymal stem cells 5,486,359 Mesenchymalstem cells 5,827,735 Mesenchymal stem cells 5,908,782 Mesenchymal stemcells 6,936,281 Mesenchymal stem cells 6,908,764 Mullerian duct-derivedpluripotent 6,416,999 epithelial cells Myeloid progenitor cells6,465,247 c-kit^(hi), IL-7Rα⁻ Myeloid progenitor cells 6,761,883 Thy-1⁻,IL-7Rα⁻ Neural progenitor cells 5,753,505 Neural stem cells 5,851,832Neuronal progenitor cells 6,251,669 Neuronal stem cells 6,969,608Neuronal progenitor cells that do not 6,852,532 express a Hu proteinLineage-restricted neuronal precursor 6,734,015 cells E-NCAM⁺Neuroepithelial stem cells 7,037,702 Central nervous system neural stemcells 5,968,829 Neuronal progenitor cells 6,812,027 Neural progenitorcells 6,913,925 Central nervous system neuron-restricted 6,787,353precursor cells Ventral mesencephalon neuron progenitor 5,411,883 cellsNeural crest stem cells 5,589,376 Neural crest multipotent cellscontaining 6,890,724 RET protein Neutrophil precursor cells 5,955,357Pancreatic progenitor cells 6,436,704 Pancreatic islet progenitor cells6,753,153 Express ErbB2 Pluripotent cells 5,914,268 Pluripotent cellstransformed with a 5,874,301 HOX11 gene Peripheral blood progenitorcells 5,541,103 Renal stem cells 6,410,320 Renal stem cells 6,458,588Retinal stem cells 6,117,675 Skeletal progenitor cells 6,517,872 Stemcells 6,767,737 FGFR⁺, not ES cells Stem cells which give rise to bloodcells 5,744,347 CD34⁻, MHC-I⁻, MHC-II⁻ T lineage progenitor cells5,821,108 CD8^(int), CD4^(int), c-kit^(hi), high bcl-2; CD8^(lo), CD4¹⁰,c-kit^(hi), high bcl-2 T lymphocyte precursor cells 5,622,853 CD34⁺,CD7⁺, Leu 8⁺

This present invention relates to pharmaceutical compositions comprisinghES cells and/or their derivatives, said stem cells being free of animalproducts, feeder cells, growth factors, leukaemia inhibitory factor,supplementary mineral combinations, amino acid supplements, vitaminsupplements, fibroblast growth factor, membrane associated steel factor,soluble steel factor and conditioned media, for use in the clinicaltreatment of presently incurable or terminal diseases, conditions ordisorders. In another embodiment, the invention relates to a method oftreatment of presently incurable or terminal conditions using hES cellsand/or their derivatives via a transplantation protocol.

The phrase “free of animal products, feeder cells, growth factors,leukaemia inhibitory factor, supplementary mineral combinations, aminoacid supplements, vitamin supplements, fibroblast growth factor,membrane associated steel factor, soluble steel factor and conditionedmedia” does not exclude the trace amounts of progestin and βhCG agonistthat may be present in the pharmaceutical composition as a result of theculturing methods of the present invention. The term “animal products”refers to any non-human product.

In another embodiment, the process of this invention provides a simple,safe, broadly applicable, reproducible and efficient method fortransplantation of a pharmaceutical composition comprising hES cellsand/or their derivatives in a ready to use form and being free of feedercells and any other contamination. These cells may be derived from anyculture methodology and transplanted into the human body irrespective ofthe patient's genetic background, age, race and gender and withoutantibody-antigen reaction or graft-host rejection, without the formationof teratomas or tumors, or other debilitating side effects, without theresult of aberrant innervations and without the need for administrationof immunosuppressors for the treatment of a variety of diseases,conditions and disorders.

In contrast to other known methods, the hES cells used in thepreparation of the pharmaceutical compositions of the present inventionare grown in a culture medium which is free of animal products, feedercells, growth factors, leukaemia inhibitory factor, supplementarymineral combinations, amino acid supplements, vitamin supplements,fibroblast growth factor, membrane associated steel factor, solublesteel factor and conditioned media, and therefore do not have any sortof contamination which may interfere with the safety or efficacy of theintended clinical use.

According to the practice of the invention, hES cells and/or theirderivatives are introduced into the human body by intramuscular,intravenous, caudal, intravitreous, intrastriatal, intraparenchymal,intrathecal, epidural, retrobulbar, subcutaneous, oral, intracardiac,intracystic, intra-articular or intrathecal injection or epiduralcatheter, sub arachnoid block catheter, intravenous infusion, vianebulizer, via spray, via intravaginal routes, and/or via local eye andear drops. In another embodiment, the hES cells and/or their derivativesare administered topically or intralesionally.

Each route of administration uses a particular volume for injection andtherefore a specific range of cells from the stock solutions of hEScells and/or their derivatives as indicated in Table 1. Where cell typesare indicated (e.g., in the injection schedule tables below), theindicated cell type is the type that is predominantly present in thecell population.

TABLE 1 Route of administration Volume Cell number intramuscular 0.25 ml750,000-1.5 million intravenous 0.25 ml 750,000-1.5 million subcutaneous0.25 ml 750,000-1.5 million caudal 2 ml 6-16 million epidural 2 ml 6-16million intrathecal 2 ml 6-16 million intra-articular 2 ml 6-16 millionretrobulbar 2 ml 6-16 million epidural catheter 4-5 ml 12-40 millionintravenous infusion 0.75 ml 2.25-4.5 million nebulizer 2 ml 6-16million intravaginal 2 ml 6-16 million

In between the laboratory and the ultimate point of use, whether it be aclinic or elsewhere, the pharmaceutical compositions must be kept incold storage. In one embodiment, the pharmaceutical compositions arestored at about +4 to about −160° C. In another embodiment, thepharmaceutical compositions are stored at about −15 to about −72° C.,e.g., about −20 to about −40° C.

In one embodiment, the hES cells used in the present invention areisolated from a spare embryo, discarded from a natural in vitrofertilization cycle and donated through informed consent. In analternate embodiment, pluripotent fetal stem cells, such as thosedescribed in U.S. Published Application No. 2005/0124003 isolated fromchorionic villus, amniotic fluid and/or placenta, are used in thepresent invention.

This invention provides a method for treatment of a variety of diseases,conditions and disorders including but not limited to cancer, stroke,genetic disorders, liver disorders, nervous system disorders, vasculardisorders, skin diseases and disorders, autoimmune disorders, eyedisorders, kidney disorders, cardiac disorders, musculoskeletaldisorders, reproductive and fertility disorders, and arthritis using hEScells and/or their derivatives.

Non-limiting examples of cancers which may be treated according to thepresent invention include spinal cord tumor, breast cancer, prostatecancer, lymphoma, skin cancer, pancreatic cancer, colon cancer,melanoma, malignant melanoma, ovarian cancer, brain cancer, primarybrain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer,bladder cancer, non-small cell lung cancer, head or neck carcinoma,breast carcinoma, ovarian carcinoma, cervical cancer, metastaticlesions, lung carcinoma, small-cell lung carcinoma, Wilms' tumor,cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreaticcarcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma,genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma,myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma,endometrial carcinoma, adrenal cortex carcinoma, malignant pancreaticinsulinoma, malignant carcinoid carcinoma, choriocarcinoma, malignanthypercalcemia, cervical hyperplasia, leukemia, acute lymphocyticleukemia, chronic lymphocytic leukemia, acute myelogenous leukemia,chronic myelogenous leukemia, chronic granulocytic leukemia, acutegranulocytic leukemia, hairy cell leukemia, neuroblastoma,rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essentialthrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissuesarcoma, osteogenic sarcoma, primary macroglobulinemia, andretinoblastoma.

In one embodiment, the hES cells are not treated with a differentiatingagent since, being embryonic, the cells are programmed, and onceadministered to the subject, the cells migrate to the site of thelesion. At that site, the cells differentiate in response to in vivodifferentiation signals. At the site of the lesion, the hES cells andtheir derivatives do not proliferate indefinitely, thereby necessitatinga schedule of repeated injections. The fact that the hES cells and theirderivatives do not proliferate indefinitely eliminates the possibilityof tumorigenicity and teratoma formation.

After treatment with hES cells and/or their derivatives according to thepractice of the present invention, the patient should take rest andadopt a strict regimen of abstinence from the intake of any substancesuch as alcohol or tobacco that may impair cellular function and impedethe regeneration processes triggered by migration of the hES cells andtheir derivatives to the site of the lesion. The patient should alsoavoid taking any medications that are known to be harmful duringpregnancy, as the medications may have adverse effects on thetransplanted cells.

Use of hES Cells and their Derivatives in the Treatment of SCI

At present there are no effective cures for the treatment of SCIincluding Acute Spinal Cord Damage, Subacute Spinal Cord Damage orChronic Spinal Cord Damage, which frequently results in paraplegia,tetraplegia and quadriplegia. The transplantation of hES cells for thetreatment of SCI presents a unique opportunity to address an unmetmedical need and dramatically improve the lives of the millions ofpeople worldwide who suffer the consequences of SCI. Regaining lostfunctions of the central and autonomic nervous systems through thetransplantation of hES cells and their derivatives in a substantiallyprogenitor state will allow recovery of parasympathetic, sympathetic,motor, autonomic and sensory pathways through the replacement of lostcell function, regeneration of lost neural cells, removal of physicalbarriers such as scar tissue, blocking of inhibitory signaling pathwaysand the release of neurotrophic factors from the transplanted hES cellprogenitors.

Amongst the benefits of restoration of neurological function through thetransplantation of hES cells according to the practice of the presentinvention are: an improvement in motor and sensory function, the generalquality of life, reduction in the support system generally provided byrelatives, decrease in dependency upon other pharmaceuticalcompositions, and other medical devices and aids, improvement in themental and psychological status and also in economic independence. Thereis a reduction in costs of medical equipment and disability aids andmedication dependence through a recovery of the parasympathetic,sympathetic, sensory, and motor functions. There is an enhancement inself-sufficiency, improvement in social status, marital status andability to exercise reproductive rights, with only a limited number ofclinic visits per annum for treatment. Treatment by the administrationof hES cells and their derivatives according to the present inventiondoes not lead to problems of antigenicity, tumour formation, teratomaformation or aberrant neural connections.

Also, administration of hES cells and their derivatives especially forSCI subjects results in the cure of bed sores, reduction in their needfor hypertensives or anti-depressants, restoration of bladder sensationand control, restoration of bowel sensation and sensation of pain andtouch; restoration of lost reflexes; reduction in symptoms of coldsweats; reduction in the sensation of giddiness; normalization of bloodpressure and normalization of breathing with full diaphragmaticinvolvement.

A procedure for the administration of hES cells to patients sufferingSub-acute Spinal Cord Damage, or Chronic Spinal Cord Damage and for whomnatural recovery mechanisms have failed is given in detail below. Adifferent procedure for the treatment of Acute SCI as a first line oftreatment within three months of the injury according to the practice ofthe present invention is also provided in detail below.

The therapeutically effective dosage, the schedule of administration androute of administration of hES cells to be administered primarilydepends upon three factors; namely type of disorder, clinical status ofthe patient and the severity of the symptoms present During the practiceof the present invention it has been found that therapeuticallyeffective dosages and schedules are not dependent upon age, gender, bodyweight or race. The protocol for each patient is individuallyestablished based on an ongoing process of evaluation of the patient.

In one embodiment of the practice of the present invention, apharmaceutical composition containing hES cells and/or their derivativesis administered to a subject that is suffering from a presentlyincurable disorder or terminal condition or other clinical disorder asreferred to above through a series of injections in order to treat thedisorder.

Various embodiments for the treatment of clinical disorders and terminalconditions according to the present invention will now be described withreference to the following examples.

Treatment, Dosage, Schedule, Routes of Administration, Evaluation andFollow Up of Patients Suffering from SCI

In one embodiment, hES cells and/or their derivatives are administeredto a subject that has suffered from a SCI for more than three months(Sub Acute and Chronic SCI) through a series of injections in order totreat the disorder. In another embodiment, the hES derivatives arehematopoietic progenitor stem cells. In another embodiment, the hESderivatives are neuronal progenitor stem cells.

Test Dose

In one embodiment, about 750,000 to about 80 million hES cells and/ortheir derivatives, wherein said cells comprise hematopoietic progenitorstem cells and neuronal progenitor stem cells, diluted in sterile normalsaline to a final volume of about 0.25 to about 1.0 ml are tested forcontamination and viability and for count using standard protocols andthereafter are administered by subcutaneous injection as a test dose inthe forearm. Observations are made to check for anaphylactic shock, painor inflammation at the site of the injection, generalized itching,flushing or fever after five minutes, ten minutes, fifteen minutes,thirty minutes, one hour and twenty four hours. In one embodiment, theproportion of hES cells to hematopoietic progenitor stem cells andneuronal progenitor stem cells ranges from about 4:1 to about 1:4. Inanother embodiment, the proportion is about 1:1.

Priming Dose

The protocol entails the administration of a subcutaneous, intramuscularand/or intravenous priming injection of a pharmaceutical compositioncontaining about 750,000 to about 80 million hES cells and/or theirderivatives, wherein said cells comprise hematopoietic progenitor stemcells and neuronal progenitor stem cells, resuspended in a volume ofabout 0.25 ml to about 1.0 ml of sterile normal saline. The priminginjection is administered through an injection of a pharmaceuticalcomposition containing the same number of hES cells and theirderivatives daily for one week to 10 days.

Epidural Injection and Epidural Catheter

About 750,000 to 80 million hES cells and/or their derivatives, whereinsaid cells comprise neuronal progenitor stem cells, suspended in avolume of 2 ml of sterile normal saline and further diluted to 5 ml to40 ml of sterile normal saline is administered by epidural injection orepidural catheter above or below the site of the lesion twice daily overa period of three consecutive days, seven to ten days after the firstpriming injection. Administration by epidural injection/epiduralcatheter is repeated above or below the site of the lesion according tothe clinical progress in the improvement of symptoms presented by thepatient and the opinion of the physician. It has also been observed thatif the patient is made to lie on his/her back after the epiduralinjection, sensory improvement is substantial and if he/she is made tolie in the face-down position, the motor improvement is substantial. Inboth cases, the patient has to have the legs kept in an elevatedposition.

Intrathecal

About 750,000 to 11 million hES cells and/or their derivatives, whereinsaid cells comprise neuronal stem cell progenitors, suspended in 2 ml ofsterile normal saline and further diluted by 2 ml of sterile normalsaline to a total volume of 4 ml are administered by intrathecalinjection above or below the site of lesion at periods of two, five,eight, twelve, seventeen and twenty two months after the start of thepriming injections.

In one embodiment, the SCI treatment is continued by administering cellsby epidural injection via catheter above or below the site of thelesion, e.g., fifteen days after the injury. In one embodiment, asuspension of about 750,000 to 80 million hES cells and/or theirderivatives, wherein said cells comprise neuronal stem cell progenitors,in a volume of 2 ml of sterile normal saline and then further dilutedinto 15 ml to 40 ml of sterile normal saline is injected. This treatmentmay be repeated one and a half months later.

Deep Spinal Injection

According to the observed progress in symptom reversal, additionalbooster injections of the pharmaceutical composition may be administeredcomprising about 750,000 to about 80 million hES cells and/or theirderivatives, wherein said cells comprise both hematopoietic stem cellsand neuronal progenitor stem cells, suspended in a volume of 0.25 ml to1.0 ml of sterile normal saline. In one embodiment, the pharmaceuticalcomposition is administered by deep spinal injection at the back of thespine weekly or every other week. This treatment will strengthen theback muscles and enhance physical rehabilitation once the patient hasregained mobility.

Caudal Injection

According to the practice of the present invention, a pharmaceuticalcomposition of about 750,000 to about 80 million hES cells and/or theirderivatives, wherein said cells comprise neuronal stem cell progenitors,resuspended in 2 ml of saline and diluted into 10 ml alone or incombination with up to 20 ml of DEPOMEDROL (methylprednisolone acetate)are administered by caudal injection. This treatment will strengthen themuscles of the lumbar region and allow for regaining of sensory andmotor power in the lumbar and sacral areas.

In addition to ongoing transplantation of hES cells, the recovery of thepatient is aided by daily physiotherapy and a re-education in their useof lost motor function and instruction on maintaining a healthylifestyle that promotes cellular function and cellular regenerationprocesses.

Local Administration

In addition to the direct treatment of SCI using hES cells and/or theirderivatives according to practice of the present invention, bed soresarising as a result of long term immobilization of the patient may betreated rapidly and effectively through the topical application of apharmaceutical composition containing hES cells and/or theirderivatives.

Treatment of bed sores may be achieved by topical application of apharmaceutical composition containing about 750,000 to about 160 millionhES cells and/or their derivatives, wherein said cells comprisehematopoietic progenitor cells. Loading or priming doses are given byintravenous and intramuscular injection to allow for internal healing aswell as to promote neo-vascularisation. Alternatively, the hES cells andtheir derivatives are resuspended in 2 ml saline, diluted with 2 to 4 mlof saline and applied intralesionally.

Intravenous Infusion

According to the practice of the present invention, a pharmaceuticalcomposition comprising 750,000 to 80 million hES cells and/or theirderivatives, wherein said cells comprise hematopoietic and neuronalprogenitor cells, are resuspended in 100 ml of normal saline andadministered by the intravenous route. This treatment insures acontinuous flow of stem cells into the body and can be especially usefulif the other direct routes are not accessible due to any reason, e.g., abed sore at the site, patient too debilitated, etc.

Protocol for Treatment of Acute SCI

Rapid intervention in the treatment of Acute SCI greatly enhances thechances of survival of the patient. Furthermore, rapid intervention inthe treatment of any incurable disease, condition or disorder throughpractice of the present invention increases the chances of recovery. Ina further embodiment of the present invention, patients suffering fromAcute SCI for less than three months after spinal injury can also betreated successfully as described in detail below. A pharmaceuticalcomposition of hES cells and/or their derivatives along with aneffective physiotherapy, rehabilitation and re-education programaccording to the practice of the present invention is an effective firstline of treatment for Acute SCI.

Treatment of Acute SCI comprises all of the steps used for treatment ofSub Acute and Chronic SCI plus two additional steps. During the initialintervention for the SCI immediately after the injury occurs (e.g.,surgery), hES cells and/or their derivatives are administered directlyto the site of the injury. Follow-up treatment comprises two intrathecalinjections and one epidural injection within three months of the injury.

Clinical Evaluation and Observations

For patients that have lost motor function in their legs for an extendedperiod of time, while being treated according to the present invention,programs for their re-education in walking and other physical therapyshould be implemented as motor function returns to their legs. As afirst step in such programs, the use of walking frames and calipers hasproven effective in re-education. An additional program ofmuscularisation of the arms and upper body should be implemented inorder for the patient to be able to support their body weight unaided inthe walking The re-education programs may be reduced as the treatmentcontinues and the condition of the patient improves.

In addition to the reduction in the need for medical device support andphysical therapy, because of improvements in the sympathetic andparasympathetic symptoms of the patient, through treatment according tothe practice of the present invention, there is a reduction in therequirement for medications, including blood pressure fluctuationmedication, depression medication, bed-sore medication, medication andmedical devices for bowel and bladder complications, anti-spasticitymedications and pumps.

In addition to a reduction in the need for medications for the treatmentof symptoms directly or indirectly associated with the SCI, it isobserved that diabetics with SCI and treated according to the presentinvention show a reduction in the need for their anti-diabeticmedications.

Clinical evaluation of the progression in improvement of the symptomspresented by the patient suffering from SCI and treated according to thepresent invention is monitored regularly during the course of thetreatment and after remission. A number of physiological, sympathetic,parasympathetic, motor, autonomic and psychological parameters areassessed in order to evaluate the efficacy of the technique in reversingthe symptoms of SCI and these are described in detail below.

An examination of the referring clinician's records, including symptomdescription, site of lesion categorization, symptom progression,clinical intervention, treatments and general history of the injury ismade. Based on this examination and a thorough examination of thepatient, including a Magnetic Resonance Imaging (MRI), Electromyography(EMG), Nerve Conduction Velocity (NCV) scan and other neurologicalexaminations and investigations including a neurological examination toassess the extent of the damage and to obtain a record of the lesionprior to treatment, methodologies for the semi-quantitative descriptionof improvement after transplantation according to the practices of thepresent invention are made and are described in detail below.

Sympathetic parameters and neurological well being including the mentalstate of the patient, whether depressed or otherwise, behavioralcharacteristics, cranial nerve function and general demeanor as part ofa psychological evaluation are noted.

An evaluation of signs and symptoms linked to the neurological damage atthe site of the lesion and functioning of the autonomic nervous systemis made. This includes a neurological examination, testing the abilityto sense deep pressure, sense of touch, sensation, balance, ability tosense pain, ability to sense change in temperature, involuntarymovements, presence of cold sweats, giddiness, blood pressure, breathingdifficulty, abnormal posture whilst lying down and ability to situnaided.

An evaluation of bladder and bowel function is made. This includesbladder control, bladder stream and sensation of fullness in thebladder, bowel control, time for evacuation of the bowel and sensationin the bowel.

An evaluation of the motor function of the upper body is made in orderto assess the extent of damage to the central nervous system. Thisincludes shoulder movement, wrist and finger movement, tendon reflexes,and strength of limb movement, muscular atrophy and hand grasp.

An evaluation of the motor function of the lower body is made in orderto assess the extent of damage to the central nervous system. Thisincludes hip movement, knee movement, toe movement, tendon reflexes, andstrength of the limb, muscular atrophy and plantar response. A detailedneurological examination is also conducted at regular intervals duringthe treatment in order to monitor the progress of limb innervation.

To aid recovery of the patient during the course of clinic visits,standard physical therapy techniques are used in order to tone thepatient as motor function returns, so that they may recover use of theirlimbs and joints and become more mobile.

Proof of neural regeneration is demonstrated by restoration ofneurological function and neurological evaluation. Examples for thetreatment protocol for SCI have been provided as case studies in thepresent application.

Treatment of Developmental Disorders of the Nervous System

hES cells and/or their derivatives according to the practice of thepresent invention are administered in an amount of about 750,000 toabout 160 million cells for the treatment of developmental disorderssuch as Autism and Mental Retardation. In another embodiment, about750,000 to about 80 million cells are administered via intramuscular orintravenous routes after a test dose. The intrathecal route can also beused. In a further embodiment, intracranial transplantation can also beused. The hES cells and/or their derivatives are predominantly neuronalprogenitor cells. In another embodiment, hematopoietic and neuronalprogenitor cells are administered via intramuscular or intravenousroutes. This treatment continues over a period of a year beginning withdaily injections via intramuscular or intravenous routes for a period of3 months. Then the same injections continue once a week for the next 3-6months and then once a fortnight for the next 3 months and then once amonth according to the physician's observations. The intrathecalinjection is included in the protocol only 6-8 months after the start ofthe treatment and then also only if the patient is showing no responseto the intramuscular and intravenous injections. In another embodimentof the present invention, 750,000 to 80 million hES cells and/or theirderivatives are resuspended in 100 ml of normal saline and administeredvia intravenous infusion

Example 1

A patient diagnosed with Autism with flapping tremors, hyperactivestate, no social skills, no eye contact, pin rolling movements,inability to follow instructions, not willing to learn, showedimprovement after the administration of a pharmaceutical compositioncomprising hES cells and their derivatives including neuronal stem cellprogenitors and hematopoietic stem cell progenitors.

The schedule of injections for this patient is shown in Table 2. Forthis table and all of the ensuing tables regarding injection schedules,the cell type indicator “neuronal” refers to a cell population of hEScells that have been partially differentiated in a medium (e.g., DMEM)that promotes differentiation to neural progenitor stem cells. The cellpopulation comprises hES cells and stem cells that are predominantlyneuronal progenitor stem cells. The indicator “non-neuronal” refers to acell population of hES cells that have been partially differentiated ina medium (e.g., RPMI) that promotes differentiation to progenitor stemcells other than neuronal progenitor stem cells. The cell populationcomprises hES cells and various stem cells that are predominantly notneuronal progenitor stem cells.

TABLE 2 Route of Date administration Cell types 3/13 test dosenon-neuronal 3/14 im neuronal 3/16 im neuronal 3/17 im neuronal 3/18 imneuronal 3/20 im neuronal 3/21 im neuronal 3/22 im neuronal 3/23 imnon-neuronal 3/24 im neuronal 3/27 im neuronal 3/28 im non-neuronal 3/29im non-neuronal 3/30 im non-neuronal 3/31 im neuronal 4/3  im neuronal4/4  im neuronal 4/5  im neuronal 4/6  im neuronal 4/7  im neuronal 4/11im neuronal 4/12 im neuronal 4/13 im neuronal 4/14 im neuronal 4/17 ivneuronal 4/19 im neuronal 4/21 iv neuronal 4/24 im neuronal 4/25 imneuronal 4/28 iv neuronal 5/1  iv neuronal 5/3  iv neuronal 5/5  imneuronal 5/8  iv neuronal 5/10 im neuronal 5/12 im neuronal 5/15 ivneuronal 5/17 im neuronal 5/29 im neuronal 5/31 iv neuronal 6/2  ivnon-neuronal 6/5  im neuronal 6/7  iv neuronal 6/9  iv non-neuronal 6/12iv neuronal 6/14 iv infusion neuronal 6/16 im neuronal 6/19 imnon-neuronal 6/21 im neuronal 6/23 iv neuronal 7/3  iv neuronal 7/5  ivinfusion neuronal 7/7  im non-neuronal 7/10 im non-neuronal 7/12 imneuronal 7/14 im neuronal 7/17 iv infusion × 2 neuronal 7/18 imnon-neuronal 7/20 im non-neuronal 7/24 im non-neuronal 7/28 imnon-neuronal 7/31 im non-neuronal 8/2  im non-neuronal 8/4  imnon-neuronal 8/7  im non-neuronal 8/10 im non-neuronal 8/11 imnon-neuronal 8/14 im non-neuronal 8/18 im non-neuronal 8/21 im neuronal8/23 im non-neuronal 8/28 im non-neuronal 8/30 im non-neuronal 9/1  imnon-neuronal 9/4  im non-neuronal

Treatment of Degenerative Nervous System Disorders

hES cells and/or their derivatives, wherein said cells comprise neuronalstem cell progenitors and hematopoietic stem cell progenitors accordingto the practice of the present invention, are administered in an amountof about 750,000 to about 160 million cells for the treatment ofDegenerative Nervous System Disorders including but not limited toCortico-Basal Degeneration, Olivo Ponto Cerebellar Atrophy, Alzheimer'sDisease, Parkinson's Disease, Multiple Sclerosis, Dementia, AuditoryNerve Atrophy and Motor Neuron Disease. In another embodiment, about750,000 to about 80 million cells are administered.

While administration protocols may be varied to suit the particularpatient, a typical protocol for the treatment of degenerative nervoussystem disorders in children comprises daily intramuscular andintravenous injections in the first month, injections three times a weekduring months 2-4, weekly injections in months 5 and 6 and weeklybooster shots in months 9-12. For adults, a typical protocol involvesdaily intramuscular and intravenous injections along with administrationby epidural catheter, lumbar puncture, intrathecal, or caudal routes.This is followed by two intravenous infusions with a minimum gap of atleast 7 days. Finally, booster injections are administered once a monthfor 6 months and then once every 2 months for at least 6 months.Treatment may be continued for longer periods and may even be life longas the diseases are progressive.

In the case of progressive and degenerative disorders, stemming orstabilizing the disease progression through rapid intervention accordingto the practice of the present invention permits an increased selfdependence of the patient. It is only after the stabilizing effect thatsome improvements can be seen.

Example 2

A patient suffered from a two-year history of repetitive progressivemultiple sclerosis taking SOLUMEDROL (methylprednisolone) daily wasunable to walk, had no bladder or bowel control, and suffered frequentlyfrom respiratory disorders.

Pursuant to the treatment according to the present invention, thesubject's condition improved, the patient was able to walk and there wasa restoration of bladder and bowel control and the use of SOLUMEDROL wasreduced. The MRI has shown a 50% improvement.

The schedule of injections for this patient is shown in Table 3.

TABLE 3 Route of Date administration Cell types  7/15 test dose neuronal 7/25 im neuronal  7/26 im neuronal  7/27 im non-neuronal  7/28 imneuronal  7/29 im neuronal 8/1 im neuronal 8/2 im neuronal 8/3 imneuronal 8/4 im × 2 neuronal 8/5 im × 2 neuronal 8/9 im × 2 neuronal 8/10 im × 2 neuronal  8/11 im neuronal  8/12 im × 2 neuronal  8/16 ivneuronal im  8/22 im × 4 hES  8/24 iv neuronal im 9/2 iv neuronal 9/8 imneuronal  9/15 im neuronal and non-neuronal mixture  9/19 iv neuronal 9/22 im neuronal  9/26 im neuronal and non-neuronal mixture  9/29 imneuronal and non-neuronal mixture 10/3  im neuronal and non-neuronalmixture 10/6  im non-neuronal 10/13 iv neuronal and non-neuronal mixture10/17 im neuronal and non-neuronal mixture 10/20 iv neuronal 10/24 ivneuronal and non-neuronal mixture 10/31 iv neuronal and non-neuronalmixture 11/3  iv neuronal and non-neuronal mixture im 11/7  imnon-neuronal 11/21 iv non-neuronal 11/24 im non-neuronal  1/19 im × 2non-neuronal neuronal  1/25 im × 2 neuronal 2/2 im non-neuronal ivneuronal  2/15 im × 2 neuronal iv non-neuronal  2/23 im × 2 neuronal ivnon-neuronal 3/2 im × 2 neuronal iv non-neuronal 3/8 im × 2 non-neuronaliv  3/16 im × 2 neuronal iv non-neuronal 5/2 im × 2 non-neuronal  5/12im × 2 neuronal iv non-neuronal 6/8 im × 2 neuronal iv non-neuronal 6/16 im × 2 neuronal iv non-neuronal 7/3 im neuronal 7/4 im neuronal7/5 im neuronal 7/6 im neuronal 7/7 im neuronal  7/18 im × 2 neuronal ivnon-neuronal  7/22 im × 2 neuronal iv non-neuronal  7/24 im × 2 neuronaliv non-neuronal 8/1 im × 2 non-neuronal iv 8/4 im × 2 non-neuronal iv8/7 im × 2 non-neuronal iv  8/24 im × 2 non-neuronal iv  8/28 im × 2non-neuronal iv 9/2 im × 2 non-neuronal iv 9/6 im × 2 non-neuronal iv9/9 im × 2 non-neuronal IV

Example 3

A 60 year old man presented with ALS (motor neuron disease) two yearsago with fast deterioration in his arms and hands. There was continuoustwitching and fasciculations in his arms, hands and tongue. He could notsupinate and pronate his left arm, or lift it above his head. His rightarm could be lifted up with a jerk and he had skeletal hands.

1½ years after treatment, his hands have recovered from the skeletalframe with regeneration of hypothenar and thenar muscles of both hands.Supination and pronation is possible with both arms. He found the samedifficulty in swallowing, which has not worsened since. There is nodeterioration with his legs, breathing and speech.

The schedule of injections for this patient is shown in Table 4.

TABLE 4 Route of Date administration Cell types 8/2  im neuronal (testdose) 8/4  im neuronal non neuronal 8/5  im neuronal 8/8  im neuronal8/9  im neuronal 8/10 im neuronal 8/11 im neuronal 8/12 im neuronal 8/16im neuronal 8/17 im hES 8/22 im neuronal 8/23 im neuronal 8/24 im × 3non-neuronal 8/25 im × 3 non-neuronal 8/26 im neuronal 8/29 im neuronal8/31 im neuronal iv 9/2  im neuronal 9/5  im neuronal iv 9/7  imneuronal 9/9  im neuronal iv mixed 9/12 im neuronal iv non neuronal 9/13im neuronal iv non neuronal 9/16 im mixed 9/19 im neuronal iv mixed 9/22iv neuronal 9/23 im neuronal iv non neuronal 9/26 im mixed 9/27 im mixed9/28 im neuronal iv non neuronal 9/30 im mixed iv non neuronal 10/3  ivmixed 10/7  im non neuronal 10/10  iv neuronal 10/12  iv mixed 10/17  imneuronal iv mixed 10/20  epidural neuronal 10/21  iv neuronal 10/24  ivmixed 10/27  iv neuronal 10/28  iv mixed 10/31  iv mixed 11/2  iv mixed11/3  epidural neuronal 11/4  iv mixed 11/7  im non neuronal 11/9  imnon neuronal 11/11  im neuronal 11/14  im neuronal 11/17  im nonneuronal 11/21  im non neuronal 11/23  im non neuronal 12/13  im nonneuronal 12/19  im neuronal 12/21  im neuronal 12/23  im neuronal 12/29 im neuronal 1/3  im neuronal 1/9  im neuronal 1/12 im neuronal 1/13 ivnon neuronal 1/16 iv neuronal 1/19 im neuronal iv non neuronal 1/23epidural neuronal 1/27 im neuronal iv non neuronal 1/29 im neuronal ivnon neuronal 2/1  iv neuronal 2/3  im neuronal iv non neuronal 2/6  imneuronal iv non neuronal 2/8  iv non neuronal 2/10 iv non neuronal 2/13iv non neuronal 2/15 iv non neuronal 2/17 im neuronal iv non neuronal2/20 im neuronal iv non neuronal 2/22 im neuronal iv non neuronal 2/24im neuronal iv non neuronal 2/27 iv neuronal oral spray non neuronal3/1  im neuronal iv non neuronal 3/6  iv non neuronal 3/8  epiduralneuronal 3/9  epidural neuronal 3/10 epidural neuronal 3/11 iv neuronal3/22 im non neuronal 3/24 im non neuronal 3/27 iv non neuronal 3/29 imnon neuronal iv 3/31 im neuronal oral spray non neuronal 4/3  imneuronal iv non neuronal 4/5  im neuronal iv non neuronal 4/7  imneuronal iv non neuronal 4/10 im neuronal iv non neuronal 4/12 imneuronal iv non neuronal 4/19 epidural neuronal 4/24 iv neuronal 4/26 ivnon neuronal 4/28 im neuronal iv non neuronal 5/2  im non neuronal 5/3 im neuronal 5/5  im neuronal iv non neuronal 5/8  iv non neuronal 5/11intravenous infusion neuronal 5/12 im non neuronal 5/14 im neuronal ivnon neuronal 5/15 im neuronal iv non neuronal 5/16 im neuronal iv nonneuronal 5/17 im neuronal iv non neuronal 5/19 im neuronal iv nonneuronal 5/22 im neuronal iv non neuronal 5/24 im neuronal iv nonneuronal 5/26 im neuronal iv non neuronal 5/29 im neuronal iv nonneuronal 6/1  im neuronal iv non neuronal 6/2  im neuronal iv nonneuronal 6/5  epidural catheter neuronal 6/6  epidural catheter neuronal6/7  epidural catheter neuronal 6/12 im neuronal non neuronal 6/14 imneuronal iv non neuronal 6/16 intravenous infusion neuronal 6/21 imneuronal 6/24 im neuronal 7/3  iv neuronal 7/5  iv neuronal 7/7  ivneuronal 7/10 iv neuronal 7/12 iv neuronal 7/14 iv neuronal 7/17 iv nonneuronal 7/20 iv non neuronal 7/24 iv non neuronal 7/25 epidural(intrathecal) non neuronal 7/31 iv neuronal 8/2  iv neuronal 8/3  iv nonneuronal 8/8  iv non neuronal 8/11 iv non neuronal 8/16 iv neuronal 8/23iv neuronal 8/28 iv non neuronal 8/29 iv non neuronal 9/5  iv nonneuronal 9/6  iv non neuronal 9/8  iv non neuronal 9/13 iv non neuronal9/15 im non neuronal iv 9/18 im non neuronal iv 9/19 intravenousinfusion neuronal 9/20 intravenous infusion non neuronal 9/22 im nonneuronal iv 9/25 im non neuronal iv 9/27 im non neuronal iv 9/29 im nonneuronal iv 10/2  im non neuronal iv 10/4  im non neuronal iv 10/9  imnon neuronal iv 10/13  im non neuronal iv 10/16  im non neuronal iv10/18  im non neuronal iv 10/20  im non neuronal iv 10/23  im nonneuronal iv 10/25  iv infusion mixed 10/27  iv infusion non neuronal11/1  im non neuronal iv 11/6  im non neuronal iv 11/8  im non neuronaliv 11/10  intrathecal neuronal 11/13  im non neuronal iv 11/15  im nonneuronal iv 11/17  im neuronal iv non neuronal 11/20  im non neuronal iv11/22  im non neuronal iv 11/24  iv neuronal 11/27  im mixed iv 12/1  immixed iv 12/4  iv infusion mixed 12/6  im non neuronal iv 12/8  ivneuronal 12/11  im non neuronal iv 12/14  iv infusion non neuronal12/15  iv infusion non neuronal 12/18  im non neuronal iv 12/20  ivneuronal 12/27  im neuronal iv 12/29  im neuronal iv 1/4  im nonneuronal iv 1/8  iv infusion mixed 1/9  iv infusion mixed 1/10 ivinfusion mixed 1/12 im non neuronal iv 1/16 im non neuronal iv 1/19 imnon neuronal iv 1/22 im mixed iv

Example 4

Patient was diagnosed to be suffering from Parkinson's disease. Patientdid not respond to the standard treatment and his condition became worseas time progressed. He had a typical shuffling gait. Unilateral tremorswere present in the right arm and he was physiologically very muchdepressed. He couldn't open his eyes. Since treatment began, the patienthas shown improvement gradually and after one year of hES cell treatmentthe patient is markedly improved. The gait is normal, the tremors in thehand are minimal and psychologically he is upbeat. He opens his eyesfully. He can sign and write now, which was impossible at the start ofthe treatment. He is less dependent on others now.

The schedule of injections for this patient is shown in Table 5.

TABLE 5 Route of Date administration Cell types 1/30 im neuronal (testdose) iv 1/31 im neuronal 2/1  iv neuronal 2/2  iv non neuronal imneuronal 2/3  iv neuronal im 2/4  iv neuronal im 2/5  iv neuronal im2/6  iv non neuronal im neuronal 2/8  iv non neuronal im neuronal 2/9 iv non neuronal im neuronal 2/10 iv non neuronal im neuronal 2/11 iv nonneuronal im 2/12 iv neuronal im non neuronal 2/13 iv neuronal im nonneuronal 2/14 iv neuronal 2/15 iv neuronal im non neuronal 2/16 ivneuronal im non neuronal 2/17 iv neuronal im non neuronal 2/18 ivneuronal im non neuronal 2/19 iv neuronal im non neuronal 2/20 ivneuronal im non neuronal 2/21 iv neuronal im non neuronal 2/23 ivneuronal im non neuronal 2/25 iv neuronal im non neuronal 2/26 ivneuronal im non neuronal 2/27 iv neuronal im non neuronal 2/28 ivneuronal 3/1  iv neuronal im non neuronal 3/2  iv neuronal im nonneuronal 3/3  iv neuronal 3/4  im neuronal 3/5  iv neuronal 3/7  imneuronal 3/9  iv non neuronal 3/10 iv neuronal im non neuronal 3/11 ivneuronal im non neuronal 3/12 iv neuronal im non neuronal 3/13 ivneuronal im non neuronal 3/28 iv neuronal 3/29 iv im neuronal iv im nonneuronal 3/30 iv im neuronal im non neuronal 3/31 iv neuronal im nonneuronal 4/1  iv neuronal im non neuronal 4/2  iv neuronal im nonneuronal 4/3  iv neuronal im non neuronal 4/4  iv im neuronal im nonneuronal 4/5  iv neuronal im non neuronal 4/6  iv neuronal im nonneuronal 4/7  iv im neuronal im non neuronal 4/8  iv neuronal im nonneuronal 4/9  iv neuronal im non neuronal 4/10 iv neuronal im nonneuronal 4/11 iv neuronal im non neuronal 4/12 iv neuronal im nonneuronal 4/13 iv neuronal im non neuronal 4/14 iv neuronal im nonneuronal 4/15 iv neuronal im non neuronal 4/16 iv neuronal im nonneuronal 5/20 iv neuronal im non neuronal 5/21 infusion non neuronal7/10 iv neuronal im non neuronal 7/11 iv neuronal im non neuronal 7/12iv neuronal im non neuronal 7/13 iv neuronal im non neuronal 7/14 ivneuronal im non neuronal 7/15 iv infusion non neuronal 7/16 iv infusionnon neuronal 7/17 iv neuronal im non neuronal 7/18 iv neuronal im nonneuronal 7/19 iv neuronal im non neuronal 7/20 iv neuronal im nonneuronal 7/21 iv neuronal im non neuronal 7/22 iv infusion neuronal 7/23iv infusion neuronal 7/24 iv infusion non neuronal 7/25 iv neuronal imnon neuronal 7/26 iv neuronal im non neuronal 7/27 iv neuronal im nonneuronal 7/28 iv non neuronal im 7/29 iv infusion neuronal 7/30 imneuronal non neuronal 9/9  im non neuronal iv 9/12 im non neuronal iv9/13 iv non neuronal im 9/14 im non neuronal iv 9/15 im non neuronal iv9/16 im non neuronal iv 9/17 iv infusion neuronal 9/18 iv infusionneuronal 9/19 iv neuronal 9/20 im non neuronal iv 9/21 im non neuronaliv 9/22 im non neuronal iv 9/23 iv infusion non neuronal 9/24 ivinfusion non neuronal 9/25 iv non neuronal im 9/26 iv non neuronal im9/27 iv non neuronal im 9/28 iv non neuronal im 9/29 iv neuronal ivinfusion 9/30 iv non neuronal im 11/9  iv non neuronal im 11/10  iv nonneuronal im 11/11  iv non neuronal im 11/12  iv non neuronal im 11/14 iv infusion mixed 11/16  iv non neuronal im 11/17  iv non neuronal im11/18  iv infusion neuronal 11/19  iv infusion neuronal 11/20  iv nonneuronal im 11/21  iv non neuronal im 11/22  iv non neuronal im 11/23 iv non neuronal im 11/24  iv infusion neuronal 11/25  iv infusionneuronal 11/26  im mixed iv 11/29  iv non neuronal im 1/9  iv nonneuronal im 1/10 iv non neuronal im 1/11 iv non neuronal im 1/12 iv nonneuronal im 1/13 iv infusion mixed 1/14 iv infusion mixed 1/15 iv nonneuronal im 1/16 iv non neuronal im 1/17 iv non neuronal im 1/18 iv nonneuronal 1/19 iv non neuronal 1/20 iv infusion non neuronal 1/21 ivinfusion mixed 1/22 im mixed iv 1/23 im mixed iv 1/24 iv neuronal im1/25 iv neuronal im 1/26 iv neuronal im 1/27 iv infusion mixed 1/28 ivinfusion mixed 1/29 iv neuronal im 1/30 iv neuronal im

Treatment of Cerebral Palsy

hES cells and/or their derivatives, wherein said cells comprise neuronalstem cell progenitors and hematopoietic stem cell progenitors accordingto the practice of the present invention, are administered in an amountof about 750,000 to about 160 million cells for the treatment ofCerebral Palsy. The protocol and the dosage are the same as that for thetreatment of neurodegenerative disorders.

Example 5

A 48 years old male diagnosed at AIIMS as CP at birth had slurredspeech, shuffling gait, difficulty in swallowing, and left sidedhemiparesis. The charting of muscle tone, reflex, and power was done toconfirm the same.

Six months after treatment he has clearer speech, no difficulty inswallowing, is able to use the left side of his body and his gait hasimproved. He is able to shave with his left hand and also use his lefthand to pick up cups, etc. His balancing is better and he is able to goon long walks.

The schedule of injections for this patient is shown in Table 6.

TABLE 6 Route of Date administration Cell types 3/8  test dosenon-neuronal 3/10 iv neuronal im 3/17 iv neuronal im 4/8  iv neuronal im× 2 4/9  ivx 3 neuronal im × 3 non-neuronal 4/10 ivx 3 neuronal im × 3non-neuronal 5/10 iv neuronal im non-neuronal 5/11 im × 2 neuronal iv5/12 iv infusion × 2 neuronal 5/13 iv infusion non-neuronal 5/14 ivinfusion neuronal iv non-neuronal im 5/15 iv infusion non-neuronal 7/19iv neuronal im non-neuronal 7/20 iv infusion non-neuronal 7/21 ivinfusion × 2 non-neuronal 7/22 iv neuronal im non-neuronal 7/24 ivneuronal im non-neuronal 7/25 deep spinal neuronal 7/26 iv neuronal imnon-neuronal 7/27 iv neuronal im non-neuronal 7/28 iv non-neuronal im7/31 iv neuronal im non-neuronal 8/1  iv non-neuronal im 8/2  ivnon-neuronal im 8/3  iv non-neuronal im 8/4  iv infusion × 2non-neuronal 8/5  iv infusion non-neuronal 8/7  iv non-neuronal im 8/8 iv non-neuronal im 8/9  iv non-neuronal im 8/10 iv non-neuronal im 8/11iv non-neuronal im 8/12 iv non-neuronal im 8/14 deep spinal neuronal8/16 iv infusion neuronal 8/17 iv infusion neuronal 8/18 iv neuronal im8/19 deep spinal neuronal 8/20 iv non-neuronal im 8/21 iv non-neuronalim 8/22 iv non-neuronal im 8/24 iv infusion non-neuronal 8/25 ivinfusion non-neuronal 8/26 iv non-neuronal im

Example 6

A 3 year old baby girl was brought into the clinic with CP and lookedand behaved like a month old baby with no neck control, weak cry, noresponse and inability to suck through the bottle. She was absolutelyfloppy.

After a year and a half of treatment she has grown and looks like a 2year old baby. She has neck control, recognizes her parents, crawls onthe bed, eats normal food, smiles on recognition, and sits with support,and has taken a few steps with her mother holding her. Her gait is ascissor gait and she has also started calling out to her parents.

The schedule of injections for this patient is shown in Table 7.

TABLE 7 Route of Date administration Cell types 8/4  im neuronal (testdose) 8/5  im neuronal 8/8  im neuronal 8/9  im neuronal 8/10 imneuronal 8/11 im neuronal 8/12 im neuronal 8/16 im neuronal 8/17 imneuronal 8/18 im neuronal 8/19 im non neuronal 8/22 im mixed 8/23 immixed 8/25 im mixed 8/26 im neuronal 8/29 im neuronal 8/30 im neuronal8/31 im neuronal 9/1  im neuronal 9/2  im neuronal 9/5  im non neuronal9/6  im neuronal 9/7  im neuronal 9/8  im neuronal 9/12 im mixed 9/14 imnon neuronal 9/16 im mixed 9/19 im neuronal 9/23 im neuronal 9/26 immixed 9/28 im mixed 9/30 im mixed 10/3  im mixed 10/5  im neuronal10/10  im non neuronal 10/17  im mixed 10/19  im non neuronal 10/21  imneuronal 10/24  im mixed 10/28  im mixed 10/31  im mixed 11/4  im mixed11/7  im non neuronal 11/9  im non neuronal 11/11  im neuronal 11/14  imneuronal 11/15  im non neuronal 11/16  im non neuronal 11/18  im nonneuronal 11/22  im non neuronal 11/23  im non neuronal 11/25  m nonneuronal 11/28  im mixed 11/29  im mixed 12/2  im mixed 12/7  im nonneuronal 12/9  m non neuronal 12/12  im non neuronal 12/14  m neuronal12/16  im neuronal 12/19  im non neuronal 12/21  m neuronal 12/23  imneuronal 12/26  m non neuronal 12/30  im neuronal 1/5  im neuronal 1/11im neuronal 1/13 im neuronal 1/16 im neuronal 1/18 im neuronal 1/23 imneuronal 1/25 im neuronal 1/27 m neuronal 1/30 im neuronal 2/1  imneuronal 2/3  im neuronal 2/6  im neuronal 2/8  im neuronal 2/10 imneuronal 2/13 im neuronal 2/15 im neuronal 2/17 im non neuronal 22 Feb.2006 im neuronal 2/24 im neuronal 2/27 im neuronal 3/1  m neuronal 3/3 im neuronal 3/6  im non neuronal 3/8  im non neuronal 3/22 im nonneuronal 3/24 im neuronal 3/27 im non neuronal 3/29 im neuronal 3/31 imneuronal 4/7  im neuronal 4/10 im neuronal 4/12 im neuronal 4/14 imneuronal 4/17 im neuronal 4/19 im neuronal 4/21 im neuronal 4/24 imneuronal 4/26 im neuronal 5/1  im neuronal 5/8  im non neuronal 5/18 imneuronal 5/22 im neuronal 5/26 im neuronal 5/29 im neuronal 5/31 imneuronal 6/2  im neuronal 6/5  im neuronal 6/12 im neuronal 6/16 imneuronal 6/19 im neuronal 6/21 im neuronal 6/28 im neuronal 6/30 imneuronal 7/4  im neuronal 7/7  im neuronal 7/17 im neuronal 7/19 mneuronal 7/21 im neuronal 7/24 im neuronal 7/26 im neuronal 7/31 im nonneuronal 8/2  im non neuronal 8/4  im non neuronal 8/7  im non neuronal8/9  im non neuronal 8/11 im non neuronal 8/14 im non neuronal 8/16 imnon neuronal 8/18 im non neuronal 8/21 im non neuronal 8/23 im nonneuronal 8/25 im non neuronal 8/28 im non neuronal 8/30 im non neuronal9/1  im non neuronal 9/4  im non neuronal 9/11 im non neuronal 9/13 imnon neuronal 9/15 im non neuronal 9/20 im non neuronal 9/25 im nonneuronal 9/27 im non neuronal 10/3  im non neuronal 10/4  im nonneuronal 10/13  im non neuronal 10/16  im non neuronal 10/18  im nonneuronal 10/19  im non neuronal 10/23  im non neuronal 10/25  im nonneuronal 10/27  im non neuronal 10/30  im non neuronal 10/31  im nonneuronal 11/3  im non neuronal 11/6  im non neuronal 11/8  im nonneuronal 11/10  im non neuronal 11/13  im non neuronal 11/15  im nonneuronal 11/17  im non neuronal 11/20  im non neuronal 11/22  im nonneuronal 11/24  im non neuronal 11/27  im non neuronal 11/29  im nonneuronal 12/1  im non neuronal 12/8  im non neuronal 12/11  im nonneuronal 12/15  im non neuronal 12/26  im neuronal 12/27  im neuronal1/3  im non neuronal 1/7  im non neuronal 1/15 im neuronal 1/24 imneuronal 1/25 im neuronal 1/29 im neuronal

Example 8

An 8 year old child was diagnosed with cerebral palsy and with severemental retardation and was unable to walk on her own. She could notidentify objects and had a very low attention span. There was a lot ofdrooling and she kept her mouth open.

Following treatment, she is not drooling, her attention span hasimproved and she is looking more alert. She is walking with minimalsupport.

The schedule of injections for this patient is shown in Table 8.

TABLE 8 Route of Date administration Cell types 10/9  im non neuronal(test dose) 10/10 im non neuronal 10/11 im non neuronal × 2 10/12 im nonneuronal 10/13 im non neuronal 12/5  im non neuronal 12/6  im nonneuronal iv 12/7  im × 3 non neuronal 12/8  im non neuronal iv 12/9  imnon neuronal iv 12/10 im × 3 non neuronal 12/11 im non neuronal iv 12/12im × 3 non neuronal 12/13 im non neuronal iv 12/14 im non neuronal 12/15im non neuronal iv 12/16 im × 3 non neuronal 12/17 im non neuronal iv12/18 im non neuronal 12/19 im non neuronal iv 12/20 im × 3 non neuronal12/21 im non neuronal iv 12/22 im non neuronal iv 12/23 im non neuronaliv 12/24 im non neuronal iv neuronal 12/25 im neuronal iv 12/26 imneuronal iv mixed 12/27 im × 3 neuronal 12/28 im neuronal iv 12/29 im ×3 neuronal 12/30 im neuronal iv 12/31 im × 3 neuronal 1/1 im mixed iv1/9 iv infusion mixed  1/10 iv infusion mixed  1/11 im non neuronal iv 1/12 im non neuronal iv  1/13 im non neuronal iv  1/14 im non neuronaliv  1/15 im × 3 non neuronal  1/16 im non neuronal iv  1/17 im × 3 nonneuronal  1/18 im non neuronal  1/19 im non neuronal  1/20 im nonneuronal  1/21 im mixed  1/22 im mixed  1/23 im mixed  1/24 im neuronal 1/25 im neuronal  1/26 im mixed  1/27 im mixed  1/28 im neuronal iv 1/29 im neuronal iv  1/30 im neuronal iv  1/31 im non neuronal 1/2 ivnon neuronal 2/2 im non neuronal

Treatment of Nervous System Trauma

hES cells and/or their derivatives, wherein said cells comprise neuronalstem cell progenitors and hematopoietic stem cell progenitors accordingto the practice of the present invention, are administered in an amountof about 750,000 to about 160 million cells for the treatment of NervousSystem Trauma, including but not limited to Brain Damage, Coma andVegetative State. In another embodiment, about 750,000 to about 80million cells are administered.

While administration protocols may be varied to suit the particularpatient, a typical protocol for the treatment of nervous system traumacomprises daily intramuscular and intravenous injections for the first 2months along with intrathecal and epidural injections.

Treatment of Cerebro-Vascular Accident or Stroke

hES cells and/or their derivatives, wherein said cells comprise neuronalstem cell progenitors and hematopoietic stem cell progenitors accordingto the practice of the present invention, are administered in an amountof about 750,000 to about 160 million cells for treatingcerebro-vascular accident or stroke. In another embodiment, about750,000 to about 80 million cells are administered.

While administration protocols may be varied to suit the particularpatient, a typical protocol for the treatment of cerebro-vascularaccident comprises daily intramuscular and intravenous injections fortwo weeks and intravenous infusion for 3 days thereafter during thefirst month, intravenous infusion for 2 days every 15 days during months2 and 3, intravenous infusion for 2 days and an intrathecal injectionduring month 5, and intravenous infusion for 4 days followed byintramuscular injection for 4 days during months 8, 10, and 12.

Example 9

A patient diagnosed with Cerebro-Vascular Accident or Stroke sufferedfrom right-sided hemiparesis with drooling, difficulty in swallowing,and inability to talk or walk. In addition, the patient had cancer ofthe colon. After the treatment according to the present invention, thepatient showed signs of improvement as the speech and motor activitiesimproved, the spine straightened, and hemiparesis was cured.

The schedule of injections for this patient is shown in Table 9.

TABLE 9 Route of Date administration Cell types  8/17 test dosenon-neuronal  8/22 im hES  8/23 im × 2 neuronal hES  8/24 im × 2 hES 8/25 im hES  8/26 im × 2 neuronal  8/29 im neuronal  8/30 iv neuronal 8/31 im neuronal 9/2 im × 2 neuronal 9/5 iv non-neuronal 9/6 ivneuronal 9/7 im neuronal 9/8 iv neuronal 9/9 iv neuronal  9/12 imneuronal and non-neuronal mixture  9/13 iv neuronal  9/14 imnon-neuronal  9/19 iv neuronal  9/22 iv neuronal  9/26 im neuronal andnon-neuronal mixture  9/29 iv neuronal 10/3  im neuronal andnon-neuronal mixture 10/6  im non-neuronal 10/10 iv neuronal 10/13 ivneuronal 10/17 iv neuronal and non-neuronal mixture 10/20 ivnon-neuronal 10/24 iv neuronal and non-neuronal mixture 10/27 ivneuronal 10/31 iv neuronal and non-neuronal mixture 11/3  iv neuronaland non-neuronal mixture 11/7  im non-neuronal 11/10 iv non-neuronal11/14 im neuronal 11/17 im non-neuronal 11/21 im non-neuronal 11/24 imnon-neuronal 12/4  iv neuronal and non-neuronal mixture 12/12 ivnon-neuronal 12/19 iv neuronal 12/26 iv non-neuronal 1/4 iv neuronal 1/11 iv infusion neuronal  8/18 iv neuronal im  8/21 iv non-neuronal im 8/22 iv non-neuronal im  8/24 im non-neuronal iv 9/1 iv infusionnon-neuronal 9/2 iv infusion non-neuronal

Example 9

An 82 year old man suffered from a cerebral stroke 5 years ago andpresented with right sided hemiparesis, with facial asymmetry andslurred speech. He walked with a stick and a huge limp and was not ableeat with his right hand due to difficulty in co-ordination. He could notsit or get up on his own and dragged his feet on walking. There wasdrooling of saliva and he also could not swallow well.

Two months after treatment he is able to walk without support, sits andstands on his own, eats with his right hand, has no slurring speech andno facial asymmetry. He is able to get up from a chair. He is bendingthe affected knee on walking while maintaining balance.

The schedule of injections for this patient is shown in Table 10.

TABLE 10 Route of Date administration Cell types 11/4  im non neuronal(test dose) 11/6  im non neuronal 11/7  im neuronal 11/8  im nonneuronal 11/9  im neuronal 11/10 im neuronal 11/11 im non neuronal 11/13im non neuronal 11/14 im non neuronal 11/15 iv neuronal im 11/16 ivneuronal im 11/18 iv non neuronal im 11/22 iv infusion neuronal 11/23 ivinfusion neuronal 11/24 im neuronal 11/27 im neuronal 11/28 im nonneuronal 11/29 im neuronal 11/30 im neuronal 12/2  im non neuronal 12/4 im non neuronal 12/5  iv infusion neuronal 12/6  iv infusion mixed im12/7  im mixed 12/8  im mixed 12/9  im mixed 12/11 im mixed 12/12 immixed 12/14 im mixed iv 12/15 im mixed 12/16 im mixed 12/18 im mixed12/19 im mixed iv 12/20 im mixed 12/21 im mixed 12/22 im mixed 12/23 immixed 12/25 im neuronal 12/26 im neuronal 12/27 iv mixed 12/28 imneuronal 12/29 im neuronal 12/30 im neuronal 1/1 im mixed 1/2 im mixed1/3 iv non neuronal 1/4 im non neuronal

Treatment of Familial Nervous System Disorders

hES cells and/or their derivatives, wherein said cells comprise neuronalstem cell progenitors and hematopoietic stem cell progenitors accordingto the practice of the present invention, are administered in an amountof about 750,000 to about 160 million cells to subjects for thetreatment of Familial Nervous System Disorders, including but notlimited to Olivo Ponto Cerebral Atrophy and Huntington's Chorea. Inanother embodiment, about 750,000 to about 80 million cells areadministered. The routes of administration are intramuscular andintravenous along with intrathecal, epidural catheter and intravenousinfusion. This would most likely continue for the person's lifetime butthe first year would be an intensive program as follows-dailyintramuscular and intravenous injections for at least three monthsduring which time intrathecal, epidural catheter and intravenousinfusion are also administered. The same set of injections areadministered after a gap of one and a half months over a period of 21days. The daily intramuscular and intravenous injections are continuedand then reduced to thrice a week, twice a week, once a week, once afortnight and then monthly depending on the patients condition. Theintrathecal injection can be repeated at a 4-6 monthly gap as can theepidural catheter. Intravenous infusions can be given every two weeks,every month or every two months.

Example 10

A patient diagnosed with Sporadic Spino Cerebellar Ataxia with inabilityto turn on the bed, walk, or shift his position whilst sitting, haddifficulty in speech, had tremors and inability to pick up his neck.

After the stem cell treatment according to the practice of the presentinvention, improvements in all of the symptoms were noted and thepatient was able to balance himself and walk a few steps. The speechbecame coherent.

The schedule of injections for this patient is shown in Table 11.

TABLE 11 Route of Date administration Cell types 8/8  test dosenon-neuronal 8/9  im neuronal 8/10 im × 2 neuronal 8/11 im × 2 neuronal8/12 im × 2 neuronal 8/13 im × 2 neuronal 8/16 im × 2 neuronal 8/17 im ×2 non-neuronal 8/18 im neuronal deep spinal 8/19 im hES 8/23 im neuronalhES 8/24 im × 2 hES 8/25 im × 2 hES 8/26 im × 2 neuronal 8/29 im × 2neuronal 8/31 im × 2 neuronal 9/5  im non-neuronal iv 9/7  im neuronal9/12 im neuronal 9/15 epidural neuronal 9/16 im neuronal 9/19 ivneuronal 9/22 iv neuronal 9/28 im neuronal 9/30 iv neuronal 10/3  imneuronal iv 10/4  im non-neuronal iv neuronal and non-neuronal mixture10/5  iv neuronal 10/13  im neuronal 10/14  intra-articular neuronal10/17  iv neuronal 11/15  iv neuronal 11/18  im non-neuronal 11/21  ivnon-neuronal 11/23  im non-neuronal 12/1  im neuronal and non-neuronalmixture iv 12/6  im neuronal and non-neuronal mixture 12/8  imnon-neuronal 12/13  im non-neuronal neuronal 12/15  epidural neuronal12/16  im neuronal 12/21  im neuronal 12/22  im neuronal iv 12/25  imnon-neuronal iv 3/5  spinal non-neuronal

Example 11

A patient with familial Olivo Ponto Cerebral Atrophy was diagnosed atJohn Hopkins University. Her father, twin sister, and brother wereafflicted by the same disease. She could only talk on expiration, waswheelchair bound with continuous dribbling of urine and had to do manualevacuation of feces. She had tremors all over the body and could notbalance herself even while sitting down. There would be severe posturalhypotension.

After 2 years of treatment the patient goes to the toilet for her bodilyfunctions, talks well, has no tremors and is able to walk with support.There has been no deterioration since even though her twin sister isbedridden with the same disease. There is no postural hypotension andshe has definitely not deteriorated.

Treatment of Skin Disorders

hES cells and/or their derivatives, wherein said cells comprisehematopoietic stem cell progenitors according to the practice of thepresent invention, are administered via subcutaneous or intravenousinjection or via local or topical application in an amount of about750,000 to about 160 million cells for the treatment of Skin disorders,including but not limited to Non-Healing Ulcers, Psoriasis, PressureSores, and Sarcoidosis. In the case of skin disorders, hES cells mayalso be used topically to treat the skin disorder or damage. In anotherembodiment, about 750,000 to about 80 million cells are administered.

In one embodiment, the hES cells or differentiated skin cells andembryonal extract may be mixed with a biocompatible carrier, such as agel, ointment or paste, and applied to damaged skin or mucosal tissue toaccelerate healing as well as to heal wounds resistant to healing, suchas diabetic or decubitus ulcers. Alternatively, the cells may beprovided in a suspension or emulsion. In one embodiment, the carrieralso contains antimicrobial agents, analgesics, or other pharmaceuticalagents.

Alternatively, in a different embodiment, hES cells are grown on asterile artificial porous substrate such as muslin and applied directlyto the wound. After 12-24 hours the muslin is removed and the stem cellscontinue growing, healing the wound. Applied intralesionally, the hEScells differentiate and eventually replace the damaged cells.

Example 12

A 70 year old lady suffered a burn injury on her left ankle which didnot heal despite conventional therapy. After application of hES cells onthe burn site, her wound started healing and three years later the skinis absolutely alright.

Treatment of Auto Immune Disorders

hES cells and/or their derivatives, wherein said cells comprisehematopoietic stem cell progenitors according to the practice of thepresent invention, are administered via intramuscular injection,intravenous injection, subcutaneous injection, intra-articular injectionor intravenous infusion or combinations thereof in an amount of about750,000 to about 160 million cells for the treatment of Auto-ImmuneDisorders including but not limited to Systemic Lupus Erythematosus,Ankylosing Spondylitis, Cardiomyopathy, Sarcoidosis, Arthritis, andUlcerative Colitis. In another embodiment, about 750,000 to about 80million cells are administered. The administration of the hES cells ishighly effective not only in the treatment of autoimmune disorders butalso in the restoration of a suppressed immune system. As an alternativeto treatment with immunosuppressors or as a method for “priming”patients for organ transplant or other medical intervention, hES cellsand their derivatives may be used to allow acceptance of the organthrough reduction in host-graft rejection processes.

While administration protocols may be varied to suit the particularpatient, a typical protocol for the treatment of autoimmune disorderscomprises daily injections for the first 2 months, alternate dayinjections along with an intravenous infusion during month 3, weeklyinjections along with a 2 day continuous intravenous infusion every 15days during months 5-7 and 10-12, and booster injections every 3 monthsfor a year and then every 6 months for a year.

Example 13

A patient suffering from psoriasis for the past 26 years was wheelchairbound with hypertension, diabetes mellitus and psoriatic arthritis withhuge ulcers on her leg which were non healing and not amenable to skingrafting.

After receiving therapy for 6 months she has no ulcers and no arthritis.Her diabetes and hypertension were under control. She has startedwalking on her own and has started all activities and is receivingbooster shots for the past 1 year. She claims that she has walked forthe first time in the past 26 years and is able to do all her householdchores on her own.

Stem cell treatment was made locally to the sores and the sores healed.

Also after the treatment no further sores were detected. The scalinessand itchiness of the skin and the swelling of the legs also was reduced.The diabetic condition was also cured with controlled blood pressurethat led to a reduction in medication.

The schedule of injections for this patient is shown in Table 12.

TABLE 12 Route of Date administration Cell types 12/27 test dosenon-neuronal  1/22 im non-neuronal  2/20 im neuronal  2/23 im neuronal 2/28 m neuronal 4/1 im neuronal 4/7 im neuronal and non-neuronalmixture  4/10 im neuronal and non-neuronal mixture  4/21 m neuronal andnon-neuronal mixture  6/29 im non-neuronal 7/6 m non-neuronal  7/29 imnon-neuronal 10/17 im non-neuronal spray 11/1  im neuronal andnon-neuronal mixture spray 11/2  im neuronal and non-neuronal mixturespray 11/3  im neuronal and non-neuronal mixture spray 11/4  im × 2neuronal 11/5  iv non-neuronal spray 11/6  im non-neuronal spray 11/7 im non-neuronal spray 11/11 iv neuronal spray 11/14 iv neuronal spray11/15 im non-neuronal spray 11/16 im non-neuronal 11/21 iv non-neuronal11/23 im non-neuronal 11/29 im neuronal and non-neuronal mixture 1/4 imnon-neuronal  3/28 im non-neuronal

Treatment of Blood Disorders

Yet another embodiment of method according to the practice of thepresent invention is in the treatment of Blood Disorders such asThrombocytopenia, Thalassemia, Acute Myeloid Leukaemia by intravenousinjection of hES cells and/or their derivatives, wherein said cellscomprise hematopoietic stem cell progenitor cells. Cells areadministered for 10 days to 14 days and then repeated as a weekly shotfor benign conditions. For malignant conditions the injections are givendaily for 40 days and then repeated if there is a relapse.

Treatment of Genetic Disorders

Another embodiment of hES cell transplantation methods according to thepractice of the present invention is in the treatment of GeneticDisorders including but not limited to Down's Syndrome, Friedereich'sAtaxia, Huntington's Chorea, Asperger's Syndrome and SpinomuscularAtrophy

The symptoms of genetic disorders are manifested by mental retardation,musculoskeletal dysfunction and organ failure in combination or aloneand result in serious incurable debilitation and reduced lifeexpectancy. In the case of patients suffering from a genetic disorder,hES cells are administered to the patient according to the practice ofthe present invention and, once administered, differentiate to providethe patient with a population of cells expressing the missing orcompromised gene product with an ensuing restoration of intracellularhomeostasis.

While administration protocols may be varied to suit the particularpatient, a typical protocol for the treatment of Genetic Disorderscomprises daily intramuscular and intravenous injections for the firstmonth, alternate day injections during month 2, injections twice a weekduring month 3, once a week injections during months 5, 7, 9, 11, and12, and booster injections every 3 months for a year. Also intravenousinfusions are given every 15 days

Example 14

A patient with Down's syndrome showed no response to verbal commands,was not able to go up and down stairs and had low body weight as aresult of eating difficulties. The patient was unable to eat, displayeda typical wide stance, and suffered from frequent coughs and colds. Theeyes were mongoloid.

Pursuant to the treatment according to the present invention, thepatient is able to understand and carry out verbal commands, startedspeaking, and started walking up and down the stairs. DQ testing hasshown the mental age to be 1 year behind her chronological age asagainst 2 years 6 months behind within a span of less than a year.Socially, she goes to school and plays with other children. She alsofeeds herself.

The schedule of injections for this patient is shown in Table 13.

TABLE 13 Route of Date administration Cell types 7/8  test dosenon-neuronal 7/16 im neuronal 7/25 im neuronal 7/26 im neuronal 7/27 imneuronal 7/30 im neuronal 8/3  im neuronal 8/10 im neuronal 8/12 imneuronal 8/16 im neuronal 8/18 im neuronal 8/22 im hES 8/26 im neuronal8/29 im neuronal 8/31 im neuronal 9/2  im neuronal 9/6  im neuronal 9/8 im non-neuronal 9/13 im non-neuronal 9/19 im neuronal and non-neuronalmixture 9/22 im non-neuronal 9/30 im non-neuronal 10/3  im neuronal andnon-neuronal mixture 10/10  im non-neuronal 10/18  im non-neuronal10/27  im neuronal 11/7  im non-neuronal 11/8  im non-neuronal 11/9  imnon-neuronal 11/10  im non-neuronal 11/11  im neuronal 11/14  imneuronal 11/17  im non-neuronal 11/18  im non-neuronal 11/21  imnon-neuronal 11/22  im non-neuronal 11/23  im non-neuronal 11/25  imnon-neuronal 11/28  im neuronal and non-neuronal mixture 12/4  imneuronal and non-neuronal mixture 12/8  im non-neuronal 12/12  imnon-neuronal 12/15  im neuronal 12/19  im neuronal 12/27  iv neuronal1/4  im non-neuronal iv 1/11 im non-neuronal neuronal 1/19 im neuronal1/20 im non-neuronal 1/23 im non-neuronal 1/30 im neuronal 2/9  imneuronal 2/13 im neuronal 2/23 im neuronal 2/27 im neuronal 3/10 imneuronal 3/14 im neuronal 3/20 im neuronal 5/11 im neuronal 5/15 imneuronal 5/16 im neuronal 5/17 im neuronal 5/18 im neuronal 5/22 imneuronal 5/23 im neuronal 7/3  im neuronal 7/5  im neuronal 7/11 imneuronal 7/13 im neuronal 7/17 im neuronal 7/19 im neuronal 7/21 ivneuronal 7/24 im neuronal 7/28 im non-neuronal 7/31 im neuronal 8/2  imnon-neuronal 8/21 im non-neuronal 8/23 im non-neuronal 8/28 imnon-neuronal 8/30 im non-neuronal

Example 15

A 3 year old girl was diagnosed with Down's syndrome. She was not ableto talk, comprehend, or walk up and down the stairs. She did not eat onher own and her single word limited speech was very unclear.

After 8 months of treatment she understands everything, speaks threeword sentences, and recognizes colors, shapes and sizes. She is able togo up and down the stairs on her own and has started eating on her own.

The schedule of injections for this patient is shown in Table 14.

TABLE 14 Route of Date administration Cell types 1/30 im neuronal (testdose) 2/6  im neuronal 2/7  im neuronal 2/8  im neuronal 2/9  imneuronal 2/10 im non-neuronal 2/13 im neuronal 2/15 im neuronal 2/16 imneuronal 2/17 im neuronal 2/20 im neuronal 2/22 im neuronal 2/24 imnon-neuronal 2/27 im neuronal 3/1  im neuronal 3/3  im non-neuronal 3/6 im non-neuronal 3/13 im neuronal 3/15 im neuronal 3/22 im neuronal 3/24im neuronal 3/26 im neuronal 3/27 im non-neuronal 3/29 im neuronal 4/3 im neuronal 4/5  im neuronal 4/7  im neuronal 4/10 im neuronal 4/12 imnon-neuronal 4/14 im non-neuronal 4/21 im neuronal 4/24 im neuronal 4/26im neuronal 4/28 im neuronal 5/1  im neuronal 5/10 im neuronal 5/12 imneuronal 5/13 im non-neuronal 5/22 im neuronal 5/24 im neuronal 5/29 imneuronal 6/5  im neuronal 6/7  im neuronal 6/10 im neuronal 6/14 imneuronal 6/16 im neuronal 6/19 im neuronal 6/21 im neuronal 6/23 ivnon-neuronal 6/27 im neuronal 6/29 im neuronal 7/3  im neuronal 7/5  imneuronal 7/12 im neuronal 7/14 im neuronal 7/17 im neuronal 7/19 imneuronal 7/21 im neuronal 7/27 im neuronal 7/28 im non-neuronal 8/1  imnon-neuronal 8/3  im non-neuronal 8/5  im non-neuronal 8/7  imnon-neuronal 8/19 im non-neuronal 8/24 im non-neuronal 8/26 im neuronal8/28 im non-neuronal 8/31 im non-neuronal 9/2  im non-neuronal 9/7  imnon-neuronal 9/9  im non-neuronal 9/11 im non-neuronal 9/14 imnon-neuronal 9/21 im non-neuronal 9/23 im non-neuronal 9/25 imnon-neuronal 9/28 im non-neuronal 9/29 im non-neuronal 10/4  imnon-neuronal 10/7  im non-neuronal 10/10  im non-neuronal 10/14  imnon-neuronal 10/16  im non-neuronal 10/18  im non-neuronal 10/22  imnon-neuronal 10/26  im non-neuronal 10/28  im non-neuronal 11/1  imnon-neuronal 11/4  im non-neuronal 11/7  im non-neuronal 11/9  imnon-neuronal 11/13  im non-neuronal 11/16  im non-neuronal 11/17  imnon-neuronal 11/20  im non-neuronal 11/23  im non-neuronal 11/27  imnon-neuronal 11/30  im non-neuronal 12/3  im non-neuronal 12/4  im × 2non-neuronal 12/7  im non-neuronal 12/9  im non-neuronal 12/11  imnon-neuronal 12/14  im × 2 non-neuronal 12/16  im non-neuronal 12/21  imnon-neuronal 12/24  im non-neuronal 12/27  im neuronal 1/1  im mixed1/3  im non neuronal 1/6  im non neuronal 1/9  im non neuronal 1/12 imnon neuronal 1/15 im non neuronal 1/18 im non neuronal 1/22 im mixed1/25 im neuronal 1/28 im neuronal

Example 16

A 13 year old boy suffered from a genetic defect with lack ofconcentration, frequent fits and sight impairment. He had total tunnelvision. He had difficulty sitting in class and seeing the blackboardeven though he was sitting in the front row. He had delayed milestonesand if he read something he would have to read it thrice to rememberwhat he read. He also had difficulty in locating items in a room.

Since starting hES cells his sight has improved and he sits two rowsback in the classroom and can see the black board much better. He isable to remember much more and his response time to verbal commands hasimproved. He does not have fits as much as before and his medication hasbeen reduced. Reports from the ophthalmologist show that the deadenedareas in the periphery have been restored.

The schedule of injections for this patient is shown in Table 15.

TABLE 15 Route of Date administration Cell types 6/8 im neuronal (testdose) 6/9 im neuronal  6/12 im neuronal  6/13 im neuronal  6/14 im nonneuronal  6/15 im non neuronal  6/16 im neuronal  6/20 im neuronal  6/21im non neuronal  6/22 im neuronal  6/23 iv non neuronal  6/27 imneuronal  6/28 im neuronal  6/29 im neuronal 7/3 im non neuronal 7/4 imneuronal 7/5 im neuronal 7/6 im neuronal 7/7 im neuronal  7/10 imneuronal  7/11 im neuronal  7/12 im neuronal  7/13 im non neuronal  7/14im neuronal  7/17 im neuronal  7/18 im neuronal  7/20 im neuronal  7/21iv infusion neuronal  7/22 iv infusion neuronal  7/24 im neuronal  7/25im neuronal  7/26 im neuronal  7/27 im neuronal  7/28 im non neuronal 7/31 im non neuronal 8/2 im non neuronal 8/3 im non neuronal 8/4 im nonneuronal 8/7 im non neuronal 8/8 im non neuronal 8/9 im non neuronal 8/10 im × 2 non neuronal  8/11 iv infusion non neuronal  8/12 ivinfusion non neuronal  8/13 im non neuronal  8/14 im non neuronal  8/17im × 2 non neuronal  8/18 im non neuronal  8/21 im non neuronal  8/22 imnon neuronal  8/23 im non neuronal  8/24 im non neuronal  8/25 im nonneuronal  8/27 im non neuronal  8/29 im non neuronal  8/30 im nonneuronal 9/2 iv infusion non neuronal 9/3 iv infusion non neuronal 9/4im × 3 non neuronal 9/5 im non neuronal 9/6 im non neuronal 9/7 im nonneuronal 9/8 im non neuronal  9/11 im non neuronal  9/13 im non neuronal 9/14 im non neuronal  9/16 im non neuronal  9/18 im non neuronal  9/19im non neuronal  9/21 im × 2 non neuronal  9/22 iv infusion non neuronal 9/23 iv infusion non neuronal 10/3  im non neuronal 10/4  im nonneuronal 10/5  im non neuronal 10/6  im non neuronal 10/9  im nonneuronal 10/10 im non neuronal 10/11 im non neuronal 10/12 im nonneuronal 10/13 retroorbital neuronal 10/16 im non neuronal 10/17 im nonneuronal 10/18 im non neuronal 10/20 im non neuronal 10/23 im nonneuronal 10/24 im non neuronal 10/25 im non neuronal 10/26 im nonneuronal 10/27 iv infusion non neuronal 10/28 iv infusion × 3 nonneuronal 10/31 im × 2 non neuronal 11/2  im × 2 non neuronal 11/3  im ×2 non neuronal 11/6  im × 2 non neuronal 11/8  im non neuronal 11/9  imnon neuronal 11/10 im non neuronal 11/13 im non neuronal 11/14retroorbital neuronal 11/15 im non neuronal 11/16 im non neuronal 11/17im non neuronal 11/20 im non neuronal 11/21 im non neuronal 11/23 im nonneuronal 11/24 im non neuronal 11/29 im non neuronal 11/30 iv mixed12/1  iv infusion mixed 12/4  im non neuronal 12/5  iv mixed 12/6  imnon neuronal 12/7  im non neuronal 12/8  im non neuronal 12/11 im nonneuronal 12/12 im non neuronal 12/13 im non neuronal 12/14 im nonneuronal 12/18 im non neuronal 12/19 iv non neuronal 12/21 im nonneuronal 12/22 retroorbital neuronal 12/29 im neuronal 2/2 im nonneuronal

Example 17

The patient is a 32 year male suffering from Fredrick's ataxia and whosesister died of the disease. He could not stand, had fading speech,difficulty in swallowing, was wheelchair bound, and needed assistance ingoing to the toilet. He was previously treated in Germany and the U.S.with no results. His heart was dilating and functioning at 15 to 20%.

Since starting hES cells, he has shown improvement. He is able to speakmuch clearer and has no difficulty in swallowing. His heart has improvedwith all the dimensions coming back to normal and the functioningcapacity up to 58 to 60%. There has been no deterioration.

The schedule of injections for this patient is shown in Table 16.

TABLE 16 Route of Date administration Cell types 2/17 im non neuronal(test dose) 2/20 im neuronal iv non neuronal 2/21 im neuronal iv nonneuronal 2/22 im neuronal iv non neuronal 2/23 im neuronal iv nonneuronal 2/24 im neuronal iv non neuronal 2/25 im neuronal iv nonneuronal 2/26 im neuronal iv non neuronal 2/27 epidural neuronal 2/28 imneuronal iv non neuronal 3/1  im neuronal iv non neuronal 3/2  imneuronal iv non neuronal 3/3  im neuronal iv non neuronal 3/4  imneuronal iv Non neuronal 3/5  im neuronal iv non neuronal 3/6  imneuronal non neuronal 3/7  epidural (catheter) neuronal 3/8  epidural(catheter) neuronal 3/9  epidural (catheter) neuronal 3/10 epidural(catheter) neuronal 3/13 im neuronal iv non neuronal 3/14 im nonneuronal 3/16 im non neuronal 3/17 im neuronal iv non neuronal 3/18 imneuronal iv non neuronal 3/19 im neuronal iv non neuronal 3/20 imneuronal iv non neuronal 3/21 im non neuronal 3/22 im non neuronal 3/24im neuronal iv non neuronal 3/25 im non neuronal 3/26 im non neuronal3/27 im neuronal 3/28 im non neuronal 3/29 im non neuronal 3/30 im nonneuronal 3/31 im non neuronal 4/1  im non neuronal 4/2  im non neuronal4/3  im non neuronal 4/4  im neuronal iv non neuronal 4/5  im nonneuronal 4/6  im non neuronal 4/7  im non neuronal 4/8  im non neuronal4/9  im neuronal iv non neuronal 4/10 im neuronal iv non neuronal 4/11im non neuronal 4/15 im non neuronal 4/16 im non neuronal 4/18 imneuronal iv non neuronal 4/19 im neuronal iv non neuronal 4/20 imneuronal iv non neuronal 4/21 im non neuronal 4/22 im non neuronal 4/24im non neuronal 4/25 im non neuronal 4/26 iv neuronal im non neuronal4/27 iv neuronal im non neuronal 4/29 iv neuronal 4/30 iv neuronal imnon neuronal 502 im non neuronal 5/3  iv neuronal 5/5  iv neuronalepidural 5/7  iv neuronal im non neuronal 5/9  iv neuronal im nonneuronal 5/10 iv neuronal im non neuronal 5/12 iv neuronal im nonneuronal 5/13 iv neuronal im non neuronal 5/14 iv neuronal im nonneuronal 5/16 epidural (intrathecal) neuronal 5/18 iv neuronal im nonneuronal 8/26 im non neuronal iv 8/27 im non neuronal iv 8/28 iv nonneuronal iv infusion 8/29 iv non neuronal iv infusion 8/30 im nonneuronal iv 8/31 im non neuronal iv 9/4  im non neuronal iv 9/6  im nonneuronal iv 9/7  im non neuronal iv 9/9  im non neuronal iv 9/10epidural (intrathecal) neuronal 9/12 im non neuronal iv 9/14 im nonneuronal iv 9/15 epidural (catheter) neuronal, 9/16 epidural (catheter)neuronal, 9/17 epidural (catheter) neuronal 9/18 im non neuronal iv 9/19im non neuronal iv 9/20 iv non neuronal iv infusion 9/21 iv non neuronaliv infusion

Treatment of Eye Disorders

Another use of hES Cell transplantation according to the practice of thepresent invention is in the treatment of Eye Disorders including but notlimited to Optical Nerve Atrophy, Macular Degeneration, Eye Damage,Corneal Graft Rejection and Retinitis Pigmentosa through directinjection of neuronal progenitors and hES cells according to thepractice of the present invention to the retrobulbar portion of the eye,superficial area of the eye and the internal chamber of the eye.

While administration protocols may be varied to suit the particularpatient, a typical protocol for the treatment of Eye Disorders comprises10 days of intramuscular and intravenous injections followed by aretrobulbar or intravitreous injection, 15 days of intramuscular andintravenous injections followed by a retrobulbar injection, 2intravenous infusions, and a retrobulbar injection after 2 months. Atotal of 4 retrobulbar injections is given over a period of 8 months toone year before results are seen but in some cases the results are seenearlier. Intravitreous routes can be used. Also the cornea can berepaired via usage of contact lenses on which the stem cells have beengrown. Also eye drops comprising stem cells can be used.

Example 18

A patient diagnosed for Optical Nerve Atrophy had conditions whichincluded difficulty in reading small letters and not being able to seeobjects placed far away. Also dark blue and violet colours were notvisible. Vision was nil with perception of light and now is 6/24.

Pursuant to the stem cell therapy according to the present invention,i.e., hES cells and their derivatives including neuronal stem cellprogenitors administered by retrobulbar injection and hES cells andtheir derivatives including neuronal stem cell progenitors andhematopoietic stem cell progenitors according to the practice of thepresent invention is administered via local intravenous injection, orsubcutaneous injection or intramuscular injection, intravitreousinjection or topical application in an amount of about 750,000 to about160 million cells, the patient showed significant improvements. He isable to read time and can see billboards as well as watch televisionfrom a distance.

The schedule of injections for this patient is shown in Table 17.

TABLE 17 Route of Date administration Cell types 2/14 im neuronal 2/15iv neuronal non-neuronal 3/20 iv × 2 neuronal im 3/21 iv neuronalretrobulbar 4/30 iv non-neuronal 5/1  iv neuronal im 5/2  retrobulbarnon-neuronal

Treatment of Liver and Kidney Disorders

Yet another use of hES cell transplantation according to the practice ofthe present invention is in the treatment of Liver Disorders and KidneyDisorders including but not limited to End Stage Renal Nephrosis andNephrotic Syndrome through transplantation according to the practice ofthe present invention. The routes of administration are intravenous,intramuscular and via intravenous infusion.

Example 19

A patient diagnosed for nephrotic syndrome exhibited elevated urea,creatine and P protein levels. The corticomedullary differentiation wasnot maintained and the body was swollen. hES cells and their derivativesincluding hematopoietic stem cell progenitors, albumin producing stemcell progenitors and bilirubin producing stem cell progenitors accordingto the practice of the present invention were administered viaintravenous injection, or subcutaneous injection, or intramuscularinjection, or intravenous infusion in an amount of about 750,000 toabout 160 million cells. After the treatment with stem cells, the bodywas less swollen and the corticomedullary differentiation was restored.The urea, creatine and P protein levels also normalized.

The schedule of injections for this patient is shown in Table 18.

TABLE 18 Route of Date administration Cell types 8/10 test dose neuronal8/11 im neuronal 8/12 im neuronal 8/16 im hES 8/22 im hES 8/24 im hES8/25 im neuronal 8/29 im neuronal 9/1  im neuronal 9/7  im neuronal 9/8 im non-neuronal 9/12 im neuronal and non-neuronal mixture 9/19 imneuronal and non-neuronal mixture 10/3  im neuronal and non-neuronalmixture 10/10  im neuronal and non-neuronal mixture 10/11  iv neuronaland non-neuronal mixture 10/31  iv non-neuronal 11/10  im non-neuronal11/21  im non-neuronal 12/8  im non-neuronal 12/23  iv non-neuronal 2/7 im non-neuronal 8/24 im non-neuronal 8/25 im non-neuronal 9/9  imnon-neuronal

Treatment of Musculo-Skeletal Disorders

Another use of hES cell transplantation according to the practice of thepresent invention is in the treatment of Musculo-Skeletal Disordersincluding but not limited to Arthritis, Duchenne's Muscular Dystrophy,Limb Girdle Dystrophy, Spinal Muscular Atrophy and Becker's MuscularAtrophy.

While administration protocols may be varied to suit the particularpatient, a typical protocol for the treatment of Musculo-SkeletalDisorders comprises daily intramuscular or intravenous injections duringthe first month, injections twice a week during months 3, 5, 6, and 7,weekly injections during months 9-12 and booster injections every 3months. Intravenous infusions are given every 10 to 15 days and deepspinal muscular injections are given every 15 to 30 days.

Example 20

The case history of a patient diagnosed with Duchenne's MuscularDystrophy showed symptoms such as listlessness, swollen thighs and face,and positive Gower's sign, with high levels of creatine phosphokinase(CPK) at 10,000 units.

After the treatment of the patient with hES cells and their derivativesincluding neuronal stem cell progenitors and hematopoietic stem cellprogenitors alone or in combination via intravenous injection, orsubcutaneous injection, or intramuscular injection, or intravenouscatheter infusion, or epidural catheter infusion or sub arachnoid blockcatheter infusion for a predetermined period, CPK levels felldrastically to 1198 units and Gower's sign was negative.

The schedule of injections for this patient is shown in Table 19.

TABLE 19 Route of Date administration Cell types 3/18 test dosenon-neuronal 3/19 im neuronal 3/20 im × 2 neuronal 3/25 im × 2non-neuronal 3/26 im × 2 non-neuronal 3/27 im × 4 neuronal × 2non-neuronal × 2 3/28 im × 4 neuronal × 2 non-neuronal × 2 3/29 im × 4neuronal × 2 non-neuronal × 2 3/30 im × 4 neuronal × 2 non-neuronal × 23/31 im × 4 neuronal non-neuronal × 3 4/1  im × 4 neuronal × 2non-neuronal × 2 4/2  im × 3 neuronal non-neuronal × 2 4/3  im × 4neuronal × 2 non-neuronal × 2 4/4  im × 4 neuronal × 2 non-neuronal × 24/5  im × 4 neuronal × 2 non-neuronal × 2 4/6  im × 4 neuronal × 2non-neuronal × 2 4/7  im × 4 neuronal × 2 non-neuronal × 2 4/8  im × 4neuronal × 2 non-neuronal × 2 4/9  im × 4 neuronal × 2 non-neuronal × 24/10 im × 4 neuronal × 2 non-neuronal × 2 4/11 im × 4 neuronal × 2non-neuronal × 2 4/12 im × 4 neuronal × 2 non-neuronal × 2 4/13 im × 4neuronal × 2 non-neuronal × 2 4/14 im × 4 neuronal × 2 non-neuronal × 24/15 im × 4 neuronal × 2 non-neuronal × 2 4/16 im × 4 neuronal × 2non-neuronal × 2 4/17 im × 5 neuronal × 3 non-neuronal × 2 4/18 im × 3neuronal non-neuronal × 2 4/19 im × 3 neuronal × 2 non-neuronal 4/20 im× 3 neuronal non-neuronal × 2 4/21 im × 3 neuronal non-neuronal × 2 4/22im × 3 neuronal non-neuronal × 2 4/23 im × 3 neuronal × 2 non-neuronal4/24 im × 4 neuronal × 2 non-neuronal × 2 4/25 im × 3 neuronalnon-neuronal × 2 4/26 im × 2 neuronal non-neuronal 7/1  im × 2 neuronalnon-neuronal 7/2  im × 4 neuronal × 2 non-neuronal × 2 7/3  im × 4neuronal × 2 non-neuronal × 2 7/4  im × 2 neuronal non-neuronal 7/5  ivinfusion × 2 neuronal 7/6  iv infusion × 2 neuronal 7/7  im × 2 neuronal7/8  im × 2 neuronal 7/9  im × 2 neuronal non-neuronal 7/10 im × 2neuronal non-neuronal 7/11 im × 2 neuronal non-neuronal 7/12 im × 2neuronal non-neuronal 7/13 im × 2 neuronal non-neuronal 7/14 im × 2neuronal non-neuronal 7/15 im × 2 neuronal non-neuronal 7/16 im × 3neuronal 7/17 iv infusion neuronal im × 2 non-neuronal 7/18 iv infusionneuronal im × 2 non-neuronal 7/19 im × 4 neuronal × 2 non-neuronal × 27/20 im × 4 neuronal × 2 non-neuronal × 2 7/21 im × 4 neuronal × 2non-neuronal × 2 7/22 im × 4 neuronal × 2 non-neuronal × 2 7/23 iv × 2neuronal × 2 im × 4 non-neuronal × 4 7/24 im × 4 neuronal × 2non-neuronal × 2 7/25 iv neuronal × 2 im × 4 non-neuronal × 4 7/26 im ×4 neuronal × 2 non-neuronal × 2 7/27 iv neuronal × 2 im × 4 non-neuronal× 4 7/28 im × 4 neuronal non-neuronal × 3 7/29 iv non-neuronal im × 47/30 im × 4 neuronal × 2 non-neuronal × 2 7/31 iv neuronal × 2 im × 4non-neuronal × 3 8/1  im × 4 neuronal × 2 non-neuronal × 2 8/2  im × 4non-neuronal 8/3  iv infusion × 2 non-neuronal 8/4  iv infusion × 2non-neuronal 8/5  im × 4 non-neuronal 8/6  im × 4 non-neuronal 8/7  ivinfusion non-neuronal im × 2 8/8  iv infusion non-neuronal im × 2 8/9 iv infusion × 2 non-neuronal im × 2 8/10 im × 4 non-neuronal 8/11 im × 4non-neuronal 8/12 iv infusion neuronal im × 2

Example 21

A 14 year old boy diagnosed with a case of DMD was bedridden with nopower in both hands and legs and with muscular atrophy. He also hadscoliosis.

After 8 months of therapy he has developed muscles in the biceps,triceps and brachio-radials and is able to lift his arms up to the elbowlevel. He has put on weight and is growing taller without any associatedspinal scoliosis. His CPK levels, which indicate the rate of musclebreakdown, have been reduced.

The schedule of injections for this patient is shown in Table 20.

TABLE 20 Route of Date administration Cell types 4/13 im non neuronal(test dose) 4/14 im non neuronal 4/15 im neuronal 4/16 im neuronal 4/17im neuronal non neuronal 4/18 im non neuronal 4/19 im neuronal nonneuronal 4/20 im neuronal non neuronal 4/21 im neuronal non neuronal4/22 im neuronal non neuronal 4/23 im neuronal non neuronal 4/24 imneuronal non neuronal 4/25 im neuronal non neuronal 4/26 im non neuronal4/27 im non neuronal 4/28 im neuronal non neuronal 4/29 im neuronal nonneuronal 4/30 im neuronal non neuronal 5/1  im non neuronal 5/2  im nonneuronal 5/3  im neuronal non neuronal 5/4  im non neuronal 5/5  imneuronal non neuronal 5/6  im neuronal 5/7  im neuronal non neuronal5/8  im neuronal non neuronal 5/9  iv infusion neuronal im 5/13 imneuronal non neuronal 5/15 im non neuronal 5/16 im non neuronal 5/17 imneuronal non neuronal 5/18 im neuronal non neuronal 5/19 im neuronal nonneuronal 5/20 im neuronal non neuronal 5/21 im neuronal non neuronal5/22 im neuronal non neuronal 5/23 im neuronal non neuronal 5/24 imneuronal non neuronal 5/26 im neuronal non neuronal 5/27 im neuronal nonneuronal 5/28 im neuronal non neuronal 5/29 im neuronal non neuronal5/30 im neuronal non neuronal 6/1  im neuronal non neuronal 6/2  imneuronal non neuronal 6/3  im neuronal non neuronal 6/4  im neuronal nonneuronal 6/5  im neuronal non neuronal 6/6  im neuronal non neuronal6/7  im neuronal non neuronal 6/8  im neuronal non neuronal 6/9  imneuronal non neuronal 6/10 im neuronal non neuronal 6/11 im neuronal nonneuronal 6/12 im non neuronal 6/13 im neuronal non neuronal 6/14 imneuronal non neuronal 6/15 im neuronal non neuronal 6/16 im neuronal nonneuronal 6/17 im neuronal non neuronal 6/18 im neuronal non neuronal6/19 im neuronal × 2 6/20 im neuronal non neuronal 6/22 im neuronal × 26/23 iv non neuronal × 2 6/24 iv non neuronal × 2 6/25 iv non neuronal ×2 6/27 im neuronal non neuronal 6/28 im neuronal non neuronal 6/29 imneuronal non neuronal 6/30 im neuronal non neuronal 7/1  im neuronal nonneuronal 7/2  im neuronal non neuronal 7/3  im neuronal non neuronal7/4  im neuronal non neuronal 7/5  im neuronal × 2 7/6  im neuronal × 27/7  im neuronal × 2 7/8  im neuronal non neuronal 7/9  im neuronal nonneuronal 7/10 im neuronal non neuronal 7/11 im neuronal non neuronal7/12 im neuronal non neuronal 7/13 im neuronal non neuronal 7/14 imneuronal non neuronal 7/15 im neuronal non neuronal 8/18 im non neuronal× 3 8/19 im non neuronal × 2 8/20 im non neuronal × 3 8/21 im nonneuronal × 3 8/22 im non neuronal × 3 8/23 im non neuronal × 3 8/24 imnon neuronal × 3 8/25 im neuronal non neuronal × 2 8/26 im neuronal × 38/27 im non neuronal × 3 8/28 iv non neuronal 7 8/29 iv non neuronal 7 ×2 8/30 im non neuronal × 2 8/31 im non neuronal × 3 9/1  iv infusion nonneuronal 9/2  iv infusion non neuronal 9/3  im non neuronal × 3 9/4  imnon neuronal × 3 9/5  im non neuronal × 3 9/6  im non neuronal × 3 9/7 im non neuronal × 3 9/8  im non neuronal × 3 9/9  im non neuronal × 39/10 im non neuronal × 3 9/11 im non neuronal × 3 9/12 im non neuronal ×3 9/13 im non neuronal × 3 9/14 im non neuronal × 3 9/15 im non neuronal× 3 9/16 im non neuronal × 3 9/17 im non neuronal × 3 9/18 im nonneuronal × 3 9/19 im non neuronal × 3 9/20 im non neuronal × 3 9/21 imnon neuronal × 3 9/22 iv infusion non neuronal 9/23 iv infusion nonneuronal 9/24 im non neuronal × 3 9/25 im non neuronal × 3 9/26 im nonneuronal × 3 9/27 im non neuronal × 3 9/28 im non neuronal × 3 9/29 imnon neuronal × 3 9/30 im non neuronal × 3 10/1  im non neuronal × 310/2  im non neuronal × 3 10/3  im non neuronal × 3 10/4  im nonneuronal × 3 10/5  im non neuronal × 3 10/6  im non neuronal × 3 10/7 im non neuronal × 3 10/8  im non neuronal × 3 10/9  im non neuronal × 310/11  im non neuronal × 3 10/12  im non neuronal × 3 10/13  im nonneuronal × 3 10/14  im non neuronal × 3 10/15  im non neuronal × 310/16  im non neuronal × 3 10/17  im non neuronal × 3 10/18  im nonneuronal × 3 10/19  im non neuronal × 3 10/20  im non neuronal × 310/21  im non neuronal × 3 10/23  im non neuronal × 3 10/24  im nonneuronal × 3 10/25  im non neuronal × 3 10/26  im non neuronal × 310/27  im non neuronal × 3 10/28  im non neuronal × 3 10/29  im nonneuronal × 3 10/30  im non neuronal × 3 10/31  im non neuronal × 5 11/2 im non neuronal × 4 11/3  im non neuronal × 3 11/4  im non neuronal × 311/5  im non neuronal × 3 11/7  im non neuronal × 3 11/8  im nonneuronal × 2 11/9  im non neuronal 11/10  im non neuronal × 3 11/11  imnon neuronal × 3 11/12  im non neuronal × 3 11/13  im non neuronal × 311/14  im non neuronal × 3 11/15  im non neuronal × 3 11/30  im mixed ×3 12/1  im non neuronal × 3 12/2  im non neuronal × 3 12/3  im nonneuronal × 3 12/4  im non neuronal × 3 12/5  im non neuronal × 3 12/6 im non neuronal × 3 12/7  im non neuronal × 3 12/8  im non neuronal × 312/9  im non neuronal × 3 12/10  im non neuronal × 3 12/12  im nonneuronal × 3 12/13  im non neuronal × 3 12/14  im non neuronal × 312/15  im non neuronal × 3 12/16  iv infusion non neuronal 12/17  ivinfusion non neuronal × 3 im 12/18  im non neuronal 12/19  iv nonneuronal im 12/20  im non neuronal × 3 12/21  iv non neuronal × 3 im12/22  iv non neuronal × 3 12/23  im non neuronal × 3 12/24  im nonneuronal × 3 12/25  im mixed × 3 12/26  iv non neuronal × 3 im 12/27  ivmixed 12/28  im non neuronal × 3 12/29  im non neuronal × 3 12/30  imnon neuronal 12/31  im non neuronal × 3 1/1  im mixed × 3 1/2  im mixed× 3 1/3  im non neuronal × 3 1/4  im non neuronal × 3 1/5  iv infusionnon neuronal 1/6  iv infusion non neuronal 1/7  im non neuronal × 3 1/8 im non neuronal × 3 1/9  im non neuronal × 3 1/10 im non neuronal × 31/11 im non neuronal × 3 1/12 im non neuronal × 3 1/13 im non neuronal ×3 1/14 im non neuronal × 3 1/15 im non neuronal × 3 1/16 iv infusionmixed 1/17 iv infusion mixed 1/18 im non neuronal 1/19 im non neuronal1/20 im non neuronal 1/21 iv infusion mixed 1/22 im mixed 1/23 im mixed1/24 im neuronal 1/25 im neuronal 1/26 im mixed 1/27 im mixed 1/28 imneuronal × 3 1/29 iv infusion mixed 1/30 iv infusion non neuronal

Example 22

An 8 year old boy wheelchair bound due to DMD was not able to walk orstand. After 6 months of treatment his CPK level was reduced. He has notdeteriorated and is able to move his arms. He has started walking withminimal support.

The schedule of injections for this patient is shown in Table 21.

TABLE 21 Route of Date administration Cell types  4/29 im neuronal nonneuronal  4/30 im non neuronal 5/1 im neuronal non neuronal 5/3 imneuronal 5/4 im neuronal non neuronal 5/5 im neuronal non neuronal 5/6im neuronal non neuronal 5/7 im non neuronal 5/8 im neuronal nonneuronal 5/9 im neuronal non neuronal  5/10 im neuronal non neuronal 5/11 im neuronal non neuronal  5/12 im neuronal non neuronal  5/13 imneuronal non neuronal  5/14 im neuronal non neuronal  5/15 im neuronalnon neuronal  5/16 im neuronal non neuronal  5/17 im neuronal nonneuronal  5/18 im neuronal non neuronal  5/19 im neuronal non neuronal 5/20 im neuronal non neuronal  5/21 im neuronal non neuronal  5/22 imneuronal non neuronal  5/23 im neuronal non neuronal  5/24 im neuronalnon neuronal  5/25 im neuronal non neuronal  5/26 im neuronal nonneuronal  5/27 im neuronal non neuronal  5/28 im neuronal non neuronal 5/29 im neuronal non neuronal  5/30 im neuronal non neuronal 6/1 imneuronal non neuronal 6/2 im neuronal non neuronal 6/3 im neuronal nonneuronal 6/4 im neuronal non neuronal 6/5 im neuronal non neuronal 6/6im neuronal non neuronal 6/7 im neuronal non neuronal 6/8 im neuronalnon neuronal 6/9 im neuronal non neuronal 11/15 im non neuronal 11/16intravenous infusion neuronal 11/17 intravenous infusion neuronal 11/18im non neuronal 11/19 im non neuronal 11/20 im non neuronal 11/21intravenous infusion mixed 11/22 intravenous infusion mixed 11/23 im nonneuronal 11/25 im mixed 11/26 im mixed 11/27 im mixed 11/29 im nonneuronal 12/1  im non neuronal 12/2  im non neuronal 12/3  im nonneuronal 12/5  im non neuronal 12/6  im non neuronal 12/7  im nonneuronal 12/8  intravenous infusion mixed 12/9  intravenous infusion nonneuronal 12/10 im non neuronal 12/11 im non neuronal 12/12 im nonneuronal 12/15 im non neuronal 12/16 intravenous infusion non neuronal12/17 intravenous infusion non neuronal 12/18 im non neuronal 12/19 imnon neuronal iv 12/20 im non neuronal 12/21 im non neuronal 12/22 im nonneuronal 12/23 im non neuronal 12/24 im non neuronal 12/25 im neuronal12/26 im neuronal 12/27 iv mixed 12/28 im neuronal 12/29 im neuronal12/30 im neuronal 12/31 im neuronal 1/1 im neuronal 1/2 im mixed 1/4 imnon neuronal 1/5 iv non neuronal 1/6 intravenous infusion non neuronal1/7 im non neuronal 1/8 im non neuronal

Example 23

A 10 year old boy diagnosed with DMD who was able to take a few stepswith support was brought for therapy. He was not able to turn on his ownon the bed or sit on his own. He could lift his arms up to 30° tillelbow level. Following a few months of treatment, his CPK level hasstarted falling and he is able to turn on his own and can lift a mug ofwater above his head to bathe. He has not lost weight.

The schedule of injections for this patient is shown in Table 22.

TABLE 22 Route of Date administration Cell types 1/20 im non neuronal(test dose) 1/23 im non neuronal 1/24 im non neuronal iv 1/25 im nonneuronal 1/26 im neuronal non neuronal 1/27 im non neuronal 2/1  im nonneuronal 2/2  im neuronal non neuronal 2/3  im non neuronal 2/4  imneuronal non neuronal 2/5  im neuronal 2/6  im non neuronal 2/7  im nonneuronal 2/8  im neuronal non neuronal 2/10 im neuronal non neuronal2/11 im neuronal non neuronal 2/12 im neuronal non neuronal 2/14 imneuronal iv non neuronal 2/15 im neuronal non neuronal 2/16 im neuronalnon neuronal 2/17 im neuronal non neuronal 2/18 im neuronal non neuronal2/21 im neuronal non neuronal 2/22 im neuronal non neuronal 2/23 imneuronal non neuronal 2/24 im non neuronal 2/25 im neuronal 2/26 imneuronal non neuronal 2/27 im neuronal non neuronal 2/28 im neuronal nonneuronal 3/28 im neuronal non neuronal 3/29 im neuronal 3/30 im neuronalnon neuronal 4/4  im neuronal 4/5  im neuronal non neuronal 4/6  imneuronal non neuronal 4/7  im neuronal non neuronal 4/8  im neuronal nonneuronal 4/9  im neuronal non neuronal 5/16 im neuronal non neuronal5/17 im neuronal non neuronal 5/18 im neuronal non neuronal 5/19 ivinfusion non neuronal 5/20 iv infusion non neuronal 5/21 im neuronal nonneuronal 5/22 im neuronal non neuronal 5/23 im neuronal non neuronal7/27 iv infusion neuronal 7/28 iv infusion neuronal im non neuronal 7/29im neuronal non neuronal 7/30 im neuronal non neuronal 7/31 im nonneuronal 8/1  im non neuronal 8/24 im neuronal non neuronal 8/25 imneuronal non neuronal 8/26 iv infusion non neuronal m 8/27 iv infusionnon neuronal im 8/28 iv neuronal non neuronal 8/29 iv infusion nonneuronal im 8/30 im non neuronal 8/31 im non neuronal 9/1  im nonneuronal 9/2  iv infusion non neuronal im 9/3  iv infusion non neuronalim 9/4  im non neuronal 9/5  im non neuronal 11/17  iv mixed im nonneuronal 11/18  iv infusion neuronal im non neuronal 11/19  iv infusionneuronal im non neuronal 11/20  im non neuronal 11/22  iv infusionneuronal 11/23  iv mixed im non neuronal 11/24  iv mixed im non neuronal11/25  im mixed iv 11/26  im mixed iv 11/27  iv non neuronal im 11/28 iv mixed 11/29  iv mixed 11/30  im mixed 12/1  iv non neuronal im 12/2 iv non neuronal im 12/3  iv non neuronal im 12/4  iv mixed 12/5  im nonneuronal iv 1/2  im mixed 1/3  im non neuronal 1/4  im non neuronal 1/5 iv infusion non neuronal im 1/6  iv infusion mixed im non neuronal 1/7 iv infusion mixed 1/8  iv infusion mixed im non neuronal 1/9  im nonneuronal

Treatment of Cardiac Diseases and Disorders

Another use of hES cell transplantation according to the practice of thepresent invention is in the treatment of Cardiac Diseases and Disordersincluding but not limited to Restrictive Cardiomyopathy, Heart Failure,Sinus Bradycardia and Coronary Artery Disease. Some portion of thedifferentiated cells are incorporated into the patient's cardiac tissue,reproducing and repairing the damaged muscle.

While administration protocols may be varied to suit the particularpatient, a typical protocol for the treatment of Cardiac Diseases andDisorders comprises intramuscular and intravenous injections every otherday for 2 weeks and then every 3 days for the next 2 weeks, andintramuscular injections once a month along with intravenous infusionsduring months 3, 6, 10, and 12. Intracardiac injections around thedamaged area may be given during bypass surgery.

Example 23

A patient diagnosed with Sinus Corda Syndrome with Sinus Bradycardia wasadvised to have a pace-maker implanted. The patient was treatedaccording to the practice of the present invention wherein the hES cellsand their derivatives including hematopoietic stem cell progenitors viaintravenous injection, or subcutaneous injection, or intramuscularinjection or intracardiac injection or during angiography wereadministered in an amount of about 750,000 to about 160 million cells.The patient showed signs of improvement after undergoing the treatmentwith hES cells and the need for a pace-maker was eliminated.

The schedule of injections for this patient is shown in Table 22.

TABLE 22 Route of Date administration Cell types 7/6  test dosenon-neuronal 7/19 im × 2 neuronal 7/21 im × 2 neuronal 7/26 im × 2neuronal 8/4  im × 2 neuronal 8/10 im × 4 neuronal 8/26 im × 2 neuronal9/1  im × 2 neuronal 9/8  iv neuronal 9/15 im neuronal and non-neuronalmixture 9/22 iv neuronal im non-neuronal 9/28 caudal neuronal 10/13  iv× 2 neuronal and non-neuronal mixture 10/27  im × 2 neuronal andnon-neuronal mixture neuronal 11/8  im × 2 non-neuronal 3/23intra-articular in neuronal knee joint 4/14 intra-articular in neuronalknee joint 5/18 intra-articular in neuronal knee joint 6/14 caudalneuronal

Treatment of Cancerous Cells and Oncogenic Tissues

Alternatively, hES cells may be administered according to the practiceof the present invention to supplement conventional chemotherapytreatment of cancer patients. In conventional chemotherapy, cytotoxicagents are administered to destroy cancer cells. However, cytotoxicagents do not distinguish between normal cells and cancer cells, and maydestroy the patient's non-cancer cells, including the cells of thepatient's immune system. As a consequence, while undergoingchemotherapy, and for some period after the chemotherapy stops, cancerpatients are susceptible to infection due to their compromised immunesystem. By administering hematopoietic stem cells, neuronal stem cellsand hES cells to patients undergoing chemotherapy some portion of theinjected cells will differentiate into a new immune system, replacingwhite blood cells, red blood cells, platelets and other cells destroyedby chemotherapy. Also, regeneration of aplastic bone marrow as a resultof radiotherapy and chemotherapy, through regulation of the mechanics ofmitosis, and restoration of normal cellular communication pathwaysboosts the immune system and halts further deregulated mitosis. The hEScells and/or their derivatives may be administered via intravenous orintramuscular injection or direct administration into the growth. Thesecells also act on the aberrant hedgehog pathway and control it so as toallow the stopping of the excessive multiplication.

Example 24

A patient reporting with grade III adeno-carcinoma underwentchemotherapy treatment and surgery without success. Because of the onsetof multiple hepatic metastases with nodular appearance and celiac andretropancreatic nodes, the patient needed supportive care.

hES cells and their derivatives according to the practice of the presentinvention were administered in an amount of about 750,000 to about 160million cells to the patient which resulted in remarkable improvementsincluding restoration of a uniformly heterogeneous liver that wasformerly nodular and an increased ecogenic area in the lymph nodes.

The schedule of injections for this patient is shown in Table 23.

TABLE 23 Route of Date administration Cell types 3/23 test dosenon-neuronal 3/24 im non-neuronal 3/25 im non-neuronal 3/26 imnon-neuronal 3/27 im non-neuronal 3/28 iv non-neuronal 3/29 ivnon-neuronal 3/30 iv non-neuronal 3/31 iv neuronal 4/1  iv non-neuronal4/2  im non-neuronal 4/3  iv non-neuronal 4/4  iv non-neuronal 4/5  ivneuronal im × 2 non-neuronal 4/6  iv non-neuronal im neuronal 4/7  ivnon-neuronal 4/8  iv non-neuronal 4/9  iv non-neuronal 4/10 ivnon-neuronal 4/11 iv non-neuronal im neuronal 4/12 iv non-neuronal imneuronal 4/13 iv neuronal im non-neuronal 4/14 iv neuronal 4/15 ivnon-neuronal im neuronal 4/16 iv non-neuronal im neuronal 4/17 ivnon-neuronal 4/19 iv non-neuronal im neuronal 4/20 iv non-neuronal imneuronal 4/21 iv non-neuronal im neuronal 4/23 iv infusion neuronal 4/24iv non-neuronal 4/25 im non-neuronal 4/26 iv non-neuronal 4/27 imnon-neuronal 5/2  iv non-neuronal 5/3  iv non-neuronal 5/6  im neuronaliv non-neuronal 5/7  im neuronal non-neuronal 5/8  iv non-neuronal 5/12iv non-neuronal 5/16 im neuronal iv non-neuronal 5/18 iv infusionnon-neuronal 5/19 iv non-neuronal 5/20 im neuronal iv non-neuronal 5/22iv infusion non-neuronal iv × 2 5/23 iv infusion non-neuronal iv × 2

Treatment of Aphthous Ulcers/Lichen Planus

Yet another use of hES cells and/or their derivatives is in thetreatment of any ulcer in the mucosal areas of the body, such asanaphthous ulcer of the mouth. The cells are administered intravenously,intramuscularly and locally. Daily injections are given intramuscularlyor intravenously for the first 4 months and intravenous infusion every15-30 days. Local application directly or via mixing with a gel is alsodone.

Example 25

A 65 year old lady came with a tongue full of necrotic tissue andaphthous ulcers which were painful. She had difficulty in eating andswallowing and in speaking. On receiving hES cells she is better;finding it easier to swallow and speak and opening her mouth better. Shealso shows recovery with the ulcers and the necrotic tissue.

Treatment of Osteoarthritis, Arthritis, Ankylosing Joints

Yet another use of hES cells and/or their derivatives is in thetreatment of Osteoarthritis, Arthritis and Ankylosing Joints. Dailyintramuscular injections are given for priming×10 days. Intra-articularinjection of 750,000 to 80 million hES cells mixed with salumedrol isgiven and repeated 1½ to 3 months later.

Treatment of Brachial Plexus Injury

Yet another use of hES cells and/or their derivatives is in thetreatment of Brachial Plexus Injuries wherein the affected arm isparalyzed. The cells are administered intramuscularly, intravenously andinto the brachial plexus and repeated every 1½ months for a year oruntil the arm is better. Intravenous infusions are also used.

Example 26

The patient is a 26 year old male who suffered from a Brachial PlexusInjury (Lt.) hand and had lost the function of it for the last 7-8years. With stem cell treatment his left hand has become much betterwith more motor power up to the elbow and wrists. He is able to move hiswrist and the hand is not as flaccid as before. He is also able to movehis arm upwards.

Treatment of Reproductive Disorders

Another use of hES cell transplantation according to the practice of thepresent invention is the treatment of Reproductive Disorders, throughthe restoration of fertility of patients suffering from testicularatrophy, ovarian failure and azoospermia.

Example 27

Treatment of an azoospermatic patient with hES cells and theirderivatives including hematopoietic stem cell progenitors via localintramuscular injection according to the practice of the presentinvention resulted in the production of spermatozoa. Also, intravenousand intramuscular injections were used as well as direct injections intothe testes and also subcutaneous injection near the epididymis wereused.

The schedule of injections for this patient is shown in Table 24.

TABLE 24 Route of Date administration Cell types 2/1 test dosenon-neuronal 8/4 im non-neuronal 2/5 im non-neuronal 8/7 im non-neuronal2/2 im neuronal and non-neuronal mixture  7/27 im neuronal 3/6 imnon-neuronal 9/1 im neuronal 9/2 im neuronal 9/6 im neuronal 9/7 imneuronal  9/12 im neuronal  9/13 im neuronal and non-neuronal mixture 1/17 iv non-neuronal  3/27 im non-neuronal  4/26 im neuronal 5/8 imnon-neuronal  5/25 im non-neuronal  8/11 im non-neuronal  8/17 imnon-neuronal 9/7 im non-neuronal

Tissue Regeneration

hES cells may be administered according to the practice of the presentinvention for the induction of tissue regeneration, including but notlimited to muscle regeneration, the treatment of liver cirrhosis, and inthe formation of new blood vessels (neo-vascularisation) which iseffective in the treatment of degenerative diseases and in treating nonhealing ulcers.

Treatment of Diabetes

Another embodiment for the invention is the use of hES cells and/ortheir derivatives, wherein said cells comprise insulin producingprogenitor cells, in the treatment of Diabetes. Diabetic patients are atfour times higher risk of suffering a heart attack or stroke and alsohave an increased risk of multi organ degeneration and failure,including the kidneys, eyes, nervous system and general immunity. Bytreatment of diabetes through the transplantation of insulin producinghES cells, and restoration of insulin production in the body, there is areduction in the need for insulin and a reduction in the debilitatingside effects.

While administration protocols may be varied to suit the particularpatient, a typical protocol for the treatment of Diabetes comprisesintramuscular and intravenous injections twice a week during the firstmonth and injections once a week during months 3, 6, 11, and 12.Intravenous infusions may also be given during month 6.

Example 28

The patient is a 70 year old man with diabetes and who had sufferedhyperketoacidosis and was on 52 units of insulin and an oralhypoglycemic. Within 6 months of therapy he was off all medications andinsulin and is now currently able to maintain his blood sugar on anormal diet for the past 1½ years. He receives booster shots.

The schedule of injections for this patient is shown in Table 25.

TABLE 25 Route of Date administration Cell types 9/5 test dosenon-neuronal 9/6 im neuronal 9/7 im neuronal 9/8 im non-neuronal 9/9 imnon-neuronal  9/12 iv neuronal  9/13 im non-neuronal  9/19 im neuronaland non-neuronal mixture  9/22 iv non-neuronal  9/26 im neuronal andnon-neuronal mixture  9/29 im neuronal and non-neuronal mixture 10/3  imneuronal and non-neuronal mixture 10/5  im non-neuronal 10/10 imnon-neuronal 10/13 iv non-neuronal 10/17 im neuronal and non-neuronalmixture 10/20 iv non-neuronal 10/24 iv neuronal and non-neuronal mixture10/31 iv neuronal and non-neuronal mixture 11/3  iv neuronal andnon-neuronal mixture 11/7  im non-neuronal 11/10 im non-neuronal 11/14iv neuronal 11/17 im non-neuronal 11/21 im non-neuronal 12/12 ivnon-neuronal 12/19 iv neuronal 12/26 iv non-neuronal 1/4 im non-neuronal 8/18 im non-neuronal  8/21 im non-neuronal  8/22 im non-neuronal  8/24im non-neuronal  8/26 im non-neuronal

Example 29

A 54 year old man presented with uncontrolled diabetes mellitus for thepast three years and was taking insulin 42 units along with an oralhypoglycemic. His fasting blood sugar was 200 mg/dl and his postprandial sugar was 280 mg/dl with all the medications.

After taking hES cells for three weeks he has stopped taking insulin andis on a reduced dose of hypoglycemic drugs. He is feeling much betterand is more alert.

The schedule of injections for this patient is shown in Table 26.

TABLE 26 Route of Date administration Cell types 1/18 im neuronal (testdose) 1/19 im non neuronal 1/20 im non neuronal 1/21 im mixed 1/22 immixed 1/23 im mixed 1/24 im neuronal 1/25 im neuronal 1/26 im nonneuronal 1/27 im non neuronal 1/28 im neuronal 1/29 im neuronal 1/30 imneuronal 1/31 im mixed

Example 30

A 45 year old man had angina and was diabetic. He was on insulin and anoral hypoglycemic and had undergone angioplasty.

Three months later after receiving hES cell treatment he has stoppedinsulin and is on a reduced dose of oral hypoglycemic. His records ofblood sugar and medication are showing well controlled blood sugarlevels.

The schedule of injections for this patient is shown in Table 27.

TABLE 27 Route of Date administration Cell types  8/21 im non neuronal(test dose)  8/22 im non neuronal  8/23 im non neuronal  8/24 im nonneuronal  8/25 im non neuronal  8/28 im non neuronal  8/30 im nonneuronal 9/1 im non neuronal 9/4 im non neuronal 9/6 iv non neuronal 9/8im non neuronal  9/11 im non neuronal  9/13 im non neuronal  9/18 im nonneuronal  9/20 im non neuronal  9/25 im non neuronal  9/28 im nonneuronal  9/29 im non neuronal  9/30 im non neuronal 10/9  im nonneuronal 10/11 im non neuronal 10/18 im non neuronal 10/23 im nonneuronal 10/25 im non neuronal 10/31 im non neuronal 11/2  im nonneuronal 11/08 im non neuronal 11/11 iv infusion mixed 11/12 iv infusionmixed 11/13 im non neuronal 11/15 im non neuronal 11/17 im non neuronal11/18 iv infusion neuronal 11/19 iv infusion neuronal 11/20 im nonneuronal 11/22 im non neuronal 11/25 iv infusion neuronal 11/26 ivinfusion neuronal 11/27 im non neuronal 11/29 iv neuronal im nonneuronal 11/30 iv infusion neuronal 12/1  iv infusion neuronal 12/10 ivinfusion mixed 12/11 im non neuronal 12/13 im non neuronal 12/16 ivinfusion neuronal 12/17 iv infusion neuronal 12/18 im non neuronal 12/20im neuronal 12/21 im neuronal 12/22 iv neuronal 1/5 im non neuronal 1/6im non neuronal iv 1/8 im non neuronal

Treatment of Interstitial Lung Disease Example 31

A middle aged lady came to the clinic suffering from ILD (interstitiallung disease). She was in a terminal stage with SPO₂ 69% at rest. As ofnow with hES cell treatment her disease has stopped progressing. Thepatient is feeling much better overall and even her breathing hasimproved. The treatment is continuing. There are breath sounds that canbe heard on auscultation and her overall pulmonary function tests showsome improvement.

The schedule of injections for this patient is shown in Table 28.

TABLE 28 Route of Date administration Cell types 10/16 im non neuronal(test dose) 10/17 im non neuronal 10/18 im non neuronal 10/19 ivneuronal epidural non neuronal nebulisation 10/20 iv neuronalnebulisation 10/21 iv neuronal nebulisation 10/22 im neuronal iv nonneuronal nebulisation 10/23 iv neuronal nebulisation 10/24 im nonneuronal nebulisation 10/25 im mixed iv nebulisation 10/26 iv mixednebulisation 10/27 iv mixed nebulisation 10/28 iv neuronal nebulisation10/29 iv neuronal nebulisation 10/30 iv mixed nebulisation 10/31 ivmixed nebulisation 11/2  iv mixed nebulisation 11/3  iv mixednebulisation 11/4  iv mixed nebulisation 11/5  iv mixed nebulisation11/6  iv mixed nebulisation 11/7  iv mixed nebulisation 11/8  iv nonneuronal nebulisation 11/9  iv mixed nebulisation 11/10 iv non neuronalnebulisation 11/11 iv mixed nebulisation 11/12 iv mixed nebulisation11/13 iv mixed nebulisation 11/14 iv mixed nebulisation 11/15 ivneuronal nebulisation 11/16 im neuronal iv non neuronal nebulisation11/17 im non neuronal iv nebulisation 11/18 im non neuronal ivnebulisation 11/19 im non neuronal iv nebulisation 11/20 IM non neuronalIV Nebulisation 11/21 im non neuronal iv nebulisation 11/22 im nonneuronal iv nebulisation 11/23 iv neuronal nebulisation 11/24 iv nonneuronal nebulisation 11/25 iv non neuronal nebulisation 11/26 iv nonneuronal nebulisation 11/27 iv non neuronal nebulisation 11/28 im nonneuronal iv nebulisation 11/29 im non neuronal iv nebulisation 11/30 imnon neuronal iv nebulisation 12/1  iv non neuronal nebulisation 12/2  ivnon neuronal nebulisation 12/3  iv non neuronal nebulisation 12/4  ivnon neuronal nebulisation 12/5  iv non neuronal nebulisation 12/6  ivnon neuronal nebulisation 12/7  iv non neuronal nebulisation 12/8  ivnon neuronal nebulisation 12/9  iv neuronal nebulisation 12/10 ivneuronal nebulisation 12/11 iv non neuronal nebulisation 12/12 iv nonneuronal nebulisation 12/13 iv non neuronal nebulisation 12/14 iv nonneuronal nebulisation 12/15 iv non neuronal nebulisation 12/16 iv nonneuronal nebulisation 12/17 iv non neuronal nebulisation 12/18 iv nonneuronal Nebulisation 12/19 IV non neuronal nebulisation 12/20 iv nonneuronal nebulisation 12/21 iv non neuronal nebulisation 12/22 ivneuronal nebulisation non neuronal 12/23 im neuronal iv non neuronalnebulisation 12/24 im neuronal iv non neuronal nebulisation 12/25 immixed iv 12/26 iv mixed nebulisation 12/27 iv mixed nebulisation 12/28iv mixed nebulisation 12/29 im neuronal iv mixed 12/30 im neuronal ivnebulisation 12/31 im neuronal iv mixed nebulisation 1/1 im mixed ivnebulisation 1/2 im mixed iv nebulisation 1/3 im non neuronal ivnebulisation 1/4 im non neuronal iv nebulisation 1/5 im neuronal iv nonneuronal nebulisation 1/6 im mixed iv non neuronal nebulisation 1/7 immixed iv non neuronal nebulisation 1/8 im mixed iv non neuronalnebulisation 1/9 im mixed iv non neuronal nebulisation  1/10 im mixed ivnon neuronal nebulisation  1/11 im mixed iv non neuronal nebulisation 1/12 im mixed iv non neuronal nebulisation  1/13 iv mixed  1/14 immixed iv non neuronal nebulisation  1/15 im neuronal iv non neuronalnebulisation  1/16 im mixed iv non neuronal nebulisation  1/17 im mixediv non neuronal Nebulisation  1/18 IM mixed IV non neuronal nebulisation 1/19 im mixed iv non neuronal nebulisation  1/20 im non neuronal ivnebulisation  1/21 im mixed iv nebulisation  1/22 im mixed ivnebulisation  1/23 im neuronal iv mixed nebulisation  1/24 im neuronaliv mixed nebulisation  1/25 im neuronal iv mixed nebulisation  1/26 imneuronal iv mixed nebulisation  1/27 im neuronal iv mixed nebulisation 1/28 im neuronal iv mixed nebulisation  1/29 im neuronal iv mixednebulisation  1/30 im non neuronal iv nebulisation  1/31 im non neuronaliv nebulisation

New Drug Development

Still another use for hES cells is for developing and testing new drugs.

For example, hES cells may be cultured according to the practice of thepresent invention and used as a substrate for testing the targets, modeof action, uptake, metabolism, excretion, toxicity and safety of newchemical entities, drug candidates and new pharmaceuticals duringpharmaceutical research and development. In one embodiment, the presentinvention relates to methods for testing the effect of a compound on hEScells and/or their derivatives, comprising culturing hES cells and/ortheir derivatives obtained by the methods of the present invention inthe presence of the compound and determining the effect of the compoundon the cells.

Example 32

The effects of the antibiotics tetracycline and ceftriaxone on culturedhES cells was analyzed along with the patient's serum. These drugs wereintroduced separately as well as together in different concentrationsand the effects studied on the stem cells. Finally the dose of themedication and the efficacy of the drugs were determined.

Drug Delivery

Another use for hES cells and/or their derivatives is to carry drugs tothe site of an injury or disease for localized delivery. hES cellsand/or their derivatives may be incubated in the presence of a drug sothat the cells take up the drug. The loaded cells can then be deliveredlocally to the treatment site where the drug will diffuse out of thecells and treatment of the injury or disease will ensue. In anotherembodiment, the loaded cells can be delivered to a site other than thedamaged or diseased area if the damaged or diseased area is unsuitablefor direct application of the drug (e.g., the area is too damaged fordirect injection of the drug). This method of drug delivery allows forthe use of drugs that would be toxic if administered systematically to asubject. The method also allows higher concentrations of the drug to beadministered to the site than may be possible by other routes ofadministration. In a further embodiment, hES cells and/or theirderivatives can be loaded with one or more drugs that will enhance thetreatment produced by the transplantation of the cells themselves.Non-limiting examples include loading cells with antihypertensive agentsfor treatment of cardiac disease, loading cells with chemotherapeuticagents for treatment of cancer patients, and loading cells withneurotrophic factors for treatment of SCI. In one embodiment, thepresent invention relates to methods for delivering a drug to a subjectcomprising culturing hES cells and/or their derivatives obtained by themethods of the present invention in the presence of the drug, whereinthe cells take up the drug, and administering the cells to a site in thesubject, wherein the drug is delivered.

In one aspect of the invention, the culture methodologies of the presentinvention allow the exposure of the hES cells and/or their derivativesto drugs and other active agents in vitro prior to transplantation, ascompared to administering the drugs or active agents directly topatients. Exposure of the cells in vitro advantageously provides thepositive effects of the drugs (e.g., the neurotrophic effects ofvalproic acid) while avoiding the toxicity of systemic administration ofthe drug.

Spinal Cord Injury Treatment: Case Studies

Clinical treatments using hES cells and their derivatives for SCI haveshown remarkable results. Given the nature of the practice of thepresent invention, and that the patients were incurable volunteers, andthat their condition was ASIA A chronic SCI, and therefore beyond thestage at which natural neural regeneration processes are possible,double-blind or placebo controlled trials have not been carried out, inrespect of the Doctors oath. The results of the protocol aredemonstrated through the detailed examples provided as case studiesbelow.

Case Study 1

The practice of the present invention resulted in the reversal of thesymptoms of SCI in a subject suffering from SCI by transplantation ofhES cells according to the protocol described herein.

ReRo 1.3.4./000/220905/α, a 29 year old subject with a C6-C7 fractureand dislocation was declared untreatable by different medical practices.The subject had no sensation from the inter mammary area downwards, andwas unable to sit on his own, had no bladder or bowel control, was notable to move his arms and fingers and had no power or tone in his legs.The subject had developed non-healing bilateral bedsores during a periodof three years. Administration of hES cells and their derivativesaccording to the practices of the present invention was initiated underthese conditions as follows.

A pharmaceutical composition containing approximately about 750,000 toabout 80 million hES cells and their derivatives including hematopoieticstem cell progenitors and neuronal stem cell progenitors was diluted insterile normal saline to a final volume of 0.25 to 1.0 ml, karyotyped,tested for contamination, viability and count using standard protocolsand then administered by subcutaneous injection in the forearm. Thesubject was observed for anaphylactic shock, pain or inflammation at thesite of the injection, generalised itching, flushing or fever after fiveminutes, ten minutes, fifteen minutes, thirty minutes, one hour andtwenty four hours.

Treatment of the subject was by administration of a subcutaneous priminginjection of a pharmaceutical composition containing 750,000 to 80million hES cells and their derivatives, wherein said cells comprisehematopoietic stem cell progenitors and neuronal stem cell progenitors,resuspended in a volume of 0.25 ml to 1.0 ml of sterile normal saline. Afurther priming injection carrying the same number of hematopoietic stemcells and neuronal stem cells resuspended in 0.25 ml to 1.0 ml ofsterile normal saline was administered by intramuscular injection. Afinal priming injection of 750,000 to 80 million neuronal stem cellprogenitors resuspended in a volume of 0.25 ml to 1.0 ml of sterilenormal saline was administered by intravenous injection.

Direct treatment of the SCI according to the practice of the presentinvention was performed by resuspension of a pharmaceutical compositioncomprising 750,000 to 80 million hES cells and their derivatives,wherein said cells comprise neuronal stem cell progenitors, resuspendedin a volume of 2 ml of sterile normal saline and further diluted to 15ml to 40 ml of sterile normal saline and administered by epiduralinjection at the site, below the site, and above the site of the lesionseven days after the first priming injection. Treatment byadministration of epidural injection was repeated after one and a halfmonths of priming, four months after priming, and six months afterpriming.

In addition to the epidural administration, the subject was treated byintrathecal injection of a pharmaceutical composition comprising 750,000to 11 million hES cells and their derivatives, wherein said cellscomprise hematopoietic stem cell progenitors and neuronal stem cellprogenitors resuspended in 2 ml of sterile normal saline two and fivemonths after the start of the treatment.

In addition, the subject was treated with an epidural injection of apharmaceutical composition comprising 750,000 to 80 million hES cellsand their derivatives, wherein said cells comprise neuronal stem cellprogenitors, resuspended in 2 ml of sterile normal saline and furtherdiluted to a final volume of 4 ml, twice daily for three consecutivedays.

The schedule of injections for this patient is shown in Table 29.

TABLE 29 Route of Date administration Cell types 8/24 test dosenon-neuronal 8/25 im × 3 hES 8/27 im × 2 neuronal 8/28 spray for bedsore neuronal 8/29 im × 4 neuronal 9/6  im × 2 neuronal 9/8  epiduralneuronal 9/9  iv neuronal 9/10 im non-neuronal 9/14 im neuronal andnon-neuronal mixture 10/4  im non-neuronal intrathecal 10/7  imnon-neuronal deep spinal 10/15  im neuronal 10/21  im non-neuronal10/26  iv neuronal 11/7  im non-neuronal 3/1  im neuronal deep spinal ×3 3/2  im × 2 neuronal 3/3  im × 2 neuronal 3/5  im neuronal 3/6  im × 2neuronal 3/7  im × 2 neuronal 3/8  im neuronal epidural × 2 3/9 epidural catheter neuronal 3/10 epidural catheter neuronal 3/11 epiduralcatheter neuronal 3/12 deep spinal neuronal 3/13 deep spinal neuronal3/14 im neuronal 3/15 im neuronal 3/16 im neuronal 3/17 im neuronal 3/18im neuronal 3/19 im neuronal 3/20 im neuronal 3/21 im neuronal 3/22 imneuronal 3/23 im neuronal 3/24 im neuronal 3/25 im neuronal 3/26 imneuronal 3/27 im neuronal 3/28 im neuronal 3/29 im neuronal 3/30 imneuronal 3/31 im neuronal 5/1  im neuronal 5/2  im neuronal 5/3 epidural catheter neuronal 5/4  epidural catheter neuronal 5/5  epiduralcatheter neuronal 5/6  im neuronal 5/8  im non-neuronal 5/12 im neuronal

The neurological well-being of the subject was evaluated at regularintervals after the start of treatment, and a marked improvement of themental state and general hygiene of the subject was observed after twoweeks as shown in TABLE 30.

TABLE 30 Cranial Time Period Mental State Hygiene Behavior Nerves 0 dayDepressed Poor Polite Normal 3 days Depressed Poor Polite Normal 15 daysHopeful Average Polite Normal 2 months Happy Average Polite Normal 3months Hopeful Average Polite Normal 10 months Very Happy Average PoliteNormal and looks forward to life

Signs and symptoms typical of damage to the autonomous nervous system asa result of a C6-C7 fracture were evaluated during the course of thetreatment. A marked and progressive improvement in all parameterstested, including the ability to sense deep pressure, sense of touch,sensation, balance, ability to sense pain, ability to sense change intemperature, involuntary movements, presence of cold sweats, giddiness,blood pressure, breathing difficulty, abnormal posture whilst lying downand ability to sit unaided, was observed as shown in TABLE 31.

TABLE 31 Time Deep Period Pressure Touch Sensation Balance Pain Temp. 0days Inter- Lower Lower Not Upper Upper mammary border border sittingborder border of clavicle of clavicle of clavicle of clavicle 3 daysUmbilicus Arm Inter- Arm Inter- Not Inter- Inter- mammary mammarysitting mammary mammary forearm 15 days Below Xiphi- Xiphi- SittingXiphi- Xiphi- umbilicus sternum sternum sternum sternum and and andinner arm inner arm inner arm 2 months Ischial Ischial Xiphi- SittingIschial Ischial spine spine sternum very well spine spine and inner arm3 months Upper Ischial Xiphi- Sitting Ischial Ischial border spinesternum spine spine of thigh and inner arm 10 months Upper PerinealUmbilicus Sitting Perineal Perineal border well of thigh 05.06 PerinealHalfway Umbilicus Sitting Perineal Perineal Down well thighs AbnormalPosture Time Cold While lying Period Sweats Giddiness BP RespirationDown 0 days ++++ ++++ Fluctuating Diaphragmatic Unable to sit breathing3 days +++ +++ Fluctuating Diaphragmatic Unable to sit breathing 15 days++ + Stable normal Sitting stable 2 months nil + on Stable normalSitting very sitting Well 3 months nil + on Stable normal Sitting verysitting Well 10 months nil + on Stable normal Standing with standingcalipers and walking frame 05.06 nil nil Stable normal Standing withcalipers and walking frame and takes a step forward

Bladder and bowel dysfunction is commonly associated with neurologicaldamage as a result of SCI. This damage results in impaired bladdercontrol, bladder stream and sensation of fullness in the bladder, bowelcontrol, time for evacuation of the bowel and sensation in the bowel.During the course of the treatment, marked and progressive improvementsin all parameters tested were observed as a result of the treatment asshown in TABLE 32.

TABLE 32 Time Involuntary Bladder Bladder Sensation Bowel Time OfSensation period Movements Control Stream (bladder) Control Evacuation(bowel) 0 day Nil Nil Nil Nil Nil 3 hours Nil 3 days Nil Nil Nil Nil Nil3 hours Nil 15 days Nil Nil Nil Nil Nil 2.5 hours   Nil 2 months Nil NilNil Nil Nil 2 hours + 3 months Nil Nil Nil Started + 0.5 hours   ++

Changes in the motor function of the upper body, as evidence for neuralregeneration at the site of the C6-C7 lesion were evaluated during thecourse of the treatment. Marked and progressive improvements in shouldermovement, wrist and finger movement, power in the fingers, tendonreflexes, strength of limb movement, muscular atrophy and hand graspwere observed as shown in TABLE 33.

TABLE 33 Wrist Reflex of Time Shoulder Fingers Tendons Power AtrophyHand Period L R L R L R S FA W F S FA W F Grasp 0 day Shrugging Wristdrop Biceps 4 2 3 0 0 3+ 4+ 4+ Nil + Wasted Brisk B/L finger BilateralNo movement 3 days Shrugging Wrist drop Biceps 4 2 3 0 0 3+ 4+ 4+ Nil +Wasted Brisk B/L finger Bilateral No movement 15 days Shrugging Wristdrop Brisk 4 3 4 3 0 3   4   4+ Present + Better Can hold Can graspGlass Can move wrist 2 months + Wrist drop Equivocal 4 4 4 4 0 2+ 3+ 3+Can put Better food in Can grasp mouth Can move wrist 3 months +Movement Equivocal 4 4 4 4 0 2+ 3+ 3+ much better S = Shoulder; FA =Forearm; W = Wrist; F = Fingers; L = Left; R = Right Power: 0 is poor, 4is good Atrophy: 0 is good, 4 is poor

Changes in the motor function of the lower body, as evidence for neuralregeneration at the site of the C6-C7 lesion, were evaluated during thecourse of the treatment, including hip movement, knee movement, toemovement, tendon reflexes, strength of the limb, muscular atrophy andplantar response. Although there was no improvement in any of theparameters tested during the course of the study, after three months oftreatment, the subject was able to stand with the aid of a walking frameas shown in TABLE 34.

TABLE 34 Time Hip Knee Toe Reflex Power Atrophy Plantar Period L R L R LR L R L R L R L R 0 day 0 0 0 0 0 0 0 0 0 0 4+ 4+ equivocal 3 days 0 0 00 0 0 0 0 0 0 4+ 4+ equivocal 15 days 0 0 0 0 0 0 0 0 0 0 4+ 4+equivocal 2 months 0 0 0 0 0 0 0 0 0 0 4+ 4+ equivocal 3 months STOOD UPWITH WALKER FOR 10 MINUTES equivocal

Case Study 2

ReRo 1.3.4./001/051004/α001, a twenty two year old subject suffered withpost traumatic paraplegia SCI as a result of a D6-D7 fracture after afall from a second floor roof top. The subject went into a coma for twodays after the fall and regained consciousness, but developed bilateralparalysis with total sensory and motor loss of the lower half of thebody from the inter mammary region to the feet, and was unable to lifthis feet or legs and was unable to sit unaided or get up into a sittingposition. The subject was permanently bed ridden, had lost bladder andbowel control and was totally dependent upon the support of his familyalthough his upper limbs were unaffected.

Administration of a pharmaceutical composition comprising hES cells andtheir derivatives according to the practice of the present invention wasinitiated after five months of the injury. In five months time, thesubject regained bladder and bowel control, and ability to sit withoutsupport, and slide up and down by lifting the hip. Sensory perceptionrestored to the perineal level. The subject can lift himself into awalking frame unaided, and can stand with ease in the frame without theneed for knee support. The treatment resulted in the ability of thesubject to walk using the walking frame with the support of a knee braceand return to work, i.e., he has regained a regular life style.

The schedule of injections for this patient is shown in Table 35.

TABLE 35 Route of Date administration Cell types 10/5  test doseneuronal 8/17 im × 3 neuronal 8/18 im neuronal 8/19 im neuronal 9/5  imnon-neuronal 9/6  im × 2 neuronal 9/7  im neuronal 9/8  im neuronal 9/10im non-neuronal 9/13 im neuronal and non-neuronal mixture 9/14 imneuronal and non-neuronal mixture 9/15 im non-neuronal 11/6  epiduralcatheter neuronal 11/7  epidural catheter × 3 neuronal 11/8  epiduralcatheter × 3 neuronal 11/9  epidural catheter × 3 neuronal 11/10 epidural catheter × 3 neuronal 11/11  iv neuronal 11/12  im neuronal11/13  iv neuronal 11/14  iv neuronal 11/15  iv neuronal 11/16  imnon-neuronal 11/18  im non-neuronal 1/12 im neuronal 2/14 imnon-neuronal 2/15 im neuronal 2/16 im non-neuronal 2/17 im non-neuronal2/18 im neuronal 3/26 im neuronal 3/27 epidural catheter × 3 neuronal3/28 epidural catheter × 4 neuronal 3/29 epidural catheter × 2 neuronal3/30 epidural catheter × 2 neuronal 3/31 iv neuronal 5/21 im neuronal5/24 intrathecal neuronal 5/26 im neuronal 7/14 im neuronal 7/15 imneuronal 7/16 im neuronal 7/17 im neuronal 7/18 intrathecal neuronal7/19 im neuronal 7/21 im neuronal 7/22 im neuronal 7/24 caudal neuronal

Case Study 3

ReRo 1.3.4./002/040205/α, a forty-year-old subject suffered fromquadriplegic SCI as a result of a C5-C6 injury and stiffness and pain inthe neck. The subject underwent surgery after six months and wasquadriplegic since then. The subject suffered from a feeling of sinkingand dizziness if made to sit with support, and had sensation from theupper border of the scapula upwards, with total loss of power in allfour limbs and loss of bowel and bladder function immediately aftersurgery.

Administration of a pharmaceutical composition comprising hES cells andtheir derivatives according to the practice of the present invention wasinitiated nine months after surgery. As a result of the hES celltreatment, the subject is able to sit comfortably without the need forsupport, and is able to move and bend to the side whilst sitting in achair with his legs hanging down comfortably. The subject regained amarked improvement of his control of his upper body. The subjectreceived substantial improvement in his general psychological well beingthrough the increased mobility and independence and activities. He isable to stand with support with strength in lower limbs, control of toemovement, and no wrist drop. Treatment is ongoing according to theimprovements of the subject's condition.

Case Study 4

ReRo 1.3.4./003/260902/β, a thirty seven year old subject who sufferedspinal injury with brain damage seventeen years ago after a road trafficaccident and was confined to a wheelchair. Subject also suffered withright-sided hemiplegia, inability to talk, facial paralysis, a totalloss of memory and no bladder control.

The subject was administered pharmaceutical composition comprising hEScells and their derivatives according to the practice of the presentinvention for one year and two months which resulted in the ability towalk with the aid of a walking frame, speaking of a few words,straightened neck, removal of facial paralysis and improving of memory.

Case Study 5

ReRo 1.3.4./004/030505/α, a fifty six year old subject who suffered frompost-traumatic fracture at C5-C8 with retrovulsion of the fracturedvertebrae causing cord contusion and associated anterior epiduralhemorrhage and was paraplegic. The subject was unable to move both lowerlimbs and suffered acute pain in his back.

The administration of a pharmaceutical composition comprising hES cellsand their derivatives according to the practice of the present inventionwas carried out for a period of one year which resulted in regaining ofsome strength in both legs, sensory perception such as vibration in thelegs, and ability to stand with the aid of a walker without back pain.The subject can walk with the aid of a walking frame, regained bladdercontrol and sensation, and overcame bouts of cold sweats and giddiness.

The schedule of injections for this patient is shown in Table 36.

TABLE 36 Route of Date administration Cell types 5/25 test dosenon-neuronal 5/27 im mixed - neuronal > hematopoietic 6/3  im mixed -neuronal > hematopoietic 6/6  im mixed - neuronal > hematopoietic 6/20im mixed - neuronal > hematopoietic 6/22 im mixed - neuronal >hematopoietic 6/24 im mixed - neuronal > hematopoietic 6/27 im mixed -neuronal > hematopoietic 6/29 iv mixed - neuronal > hematopoietic 6/30im mixed - neuronal > hematopoietic iv 7/1  iv mixed - neuronal >hematopoietic 7/4  im mixed - neuronal > hematopoietic 7/5  iv mixed -neuronal > hematopoietic 7/6  im mixed - neuronal > hematopoietic 7/7 iv mixed - neuronal > hematopoietic 7/11 im non-neuronal 7/12 im hES7/18 iv neuronal 7/19 im neuronal 7/20 im neuronal 7/21 im × 2 neuronal7/25 im × 2 neuronal 7/26 im neuronal 7/27 im × 2 neuronal 7/28 imneuronal 7/29 im × 2 neuronal 7/30 im × 2 neuronal 8/1  im × 2 neuronal8/2  im × 2 neuronal 8/16 im × 2 neuronal 8/17 im neuronal 8/25 im × 2hES 8/30 im × 2 neuronal 9/1  im × 2 neuronal 9/2  im × 2 neuronal 9/6 intrathecal mixed - neuronal > hematopoietic 9/7  im neuronal 9/13 imneuronal and non-neuronal mixture 9/14 im neuronal and non-neuronalmixture 9/15 im neuronal and non-neuronal mixture 9/19 im × 2 neuronalneuronal and non-neuronal mixture 10/4  im neuronal 10/5  im neuronalintrathecal 10/11  deep spinal neuronal 10/13  epidural neuronal 10/17 side of the spine neuronal 11/5  intrathecal neuronal 11/7  imnon-neuronal 11/8  im non-neuronal 11/9  deep spinal neuronal 11/17  im× 2 non-neuronal patient in hospital until January 1/12 im non-neuronal1/25 im neuronal 2/8  im neuronal 3/2  im × 3 neuronal (2) non-neuronal3/8  im non-neuronal 3/9  im non-neuronal 3/10 im non-neuronal 3/13epidural catheter × 4 neuronal 3/14 epidural catheter × 4 neuronal 3/15epidural catheter × 4 neuronal 3/16 epidural catheter × 2 neuronal 3/20im neuronal 3/22 im neuronal 3/28 im non-neuronal 3/29 im non-neuronal4/3  im non-neuronal 4/4  im non-neuronal 4/7  im × 2 neuronal 4/14 imneuronal 4-20 epidural catheter × 5 neuronal 4/21 epidural catheter × 3neuronal 4/24 im neuronal 5/19 im × 2 neuronal non-neuronal 5/25 caudalneuronal 6/5  iv infusion × 2 neuronal 6/6  iv infusion × 2 neuronal6/9  intrathecal neuronal 6/10 iv infusion non-neuronal 6/11 iv infusionnon-neuronal 6/12 iv non-neuronal 6/13 im non-neuronal 6/21 im neuronal7/4  im neuronal 7/10 intrathecal neuronal 7/11 iv infusion × 2 neuronal7/12 iv infusion × 2 neuronal 7/13 caudal neuronal 7/14 iv non-neuronal

Case Study 6

ReRo 1.3.4./005/130705/β, a twenty five year old subject who wasdiagnosed with Potts Spine disorder at the D6 level with lower limbparaplegia, underwent surgery three times with an anterior decompressionof the spinal cord. The subject was wheelchair bound, could not sitwithout support, with flaccid paralysis of the legs, had no bowelcontrol and evacuated whilst lying and had no bladder sensation.

The subject was administered a pharmaceutical composition comprising hEScells and their derivatives according to the practice of the presentinvention, eleven years and six months after the injury. The treatmentprotocol followed during the first year resulted in strengthening theback, ability to sit without support, and sensation of heaviness in thelegs. The subject also regained backache during menstruation andmenstrual pain. Sensations in the thighs, legs and recovery of bladdersensation and control were observed. The subject is able to walk withthe aid of a walking frame with good restoration of movement in both thelegs. Treatment is ongoing.

The schedule of injections for this patient is shown in Table 37.

TABLE 37 Route of Date administration Cell types  7/13 test dosenon-neuronal  7/27 im neuronal  7/28 im non-neuronal  7/29 im neuronal8/3 im × 2 neuronal 8/4 im × 2 non-neuronal 8/5 im neuronal 8/9 imneuronal  8/12 im × 2 neuronal  8/16 im neuronal  8/23 im × 2 hESneuronal  8/30 im × 2 neuronal 9/1 im × 4 neuronal 9/2 im × 2 neuronal9/8 iv neuronal  9/12 im neuronal  9/14 im neuronal and non-neuronalmixture  9/22 iv neuronal  9/27 im neuronal and non-neuronal mixture 9/29 im neuronal and non-neuronal mixture  9/30 im neuronal 10/4  imneuronal and non-neuronal mixture 10/6  epidural × 2 vials neuronal10/10 im neuronal 10/19 im non-neuronal 10/21 im neuronal 10/24 epiduralneuronal 11/7  epidural neuronal 11/14 iv neuronal 11/15 im non-neuronal11/18 im non-neuronal 12/1  intrathecal neuronal and non-neuronalmixture 12/7  deep spinal non-neuronal 12/23 im neuronal 12/26 imnon-neuronal 1/5 im neuronal  1/11 im neuronal  1/17 im neuronal  1/24iv neuronal  1/26 im neuronal 2/1 im neuronal 2/3 im neuronal 2/9 imnon-neuronal  2/15 im non-neuronal  2/22 im neuronal 3/1 im non-neuronal3/9 im non-neuronal  3/21 im neuronal  3/28 im neuronal  4/13 epiduralcatheter neuronal  4/26 im neuronal 5/2 im non-neuronal 5/4 epiduralcatheter neuronal 5/5 epidural catheter neuronal 5/6 epidural catheterneuronal 5/7 im neuronal  5/21 im neuronal 6/5 im neuronal  6/12 imneuronal  6/26 iv × 2 non-neuronal 7/8 im neuronal

Case Study 7

ReRo 1.3.4./006/220805/α, a thirty year old subject suffering from aC6-C7 paraplegic SCI was unable to move lower limbs and had no bowelcontrol or bowel sensation. The subject had fine and deep pressuresensation only from the inter mammary region upwards. Thus, the subjecthad difficulty in sitting. The hands of the subject had very littlestrength, with very weak finger movement and the subject had difficultyin breathing.

The subject was treated according to the practice of the presentinvention by administration of a pharmaceutical composition comprisinghES cells and their derivatives about three months after the injury. Thesubject regained ability to sit without support, suffers no giddiness,can sense pressure in the groin, has sensation in the medial side of theelbow and feels pain in the legs if the patient attempted to move. Thesubject breathes easily, has sensation in bladder and bowel, hassensation in legs and can now stand for a few minutes with support. Thesubject also has increased strength and movement in fingers. Treatmentis ongoing.

The schedule of injections for this patient is shown in Table 38.

TABLE 38 Route of Date administration Cell types 2/22 test dosenon-neuronal 2/23 im neuronal 2/24 im neuronal 2/27 im non-neuronal 2/28im non-neuronal 3/2  im neuronal 3/3  im neuronal 3/8  epidural catheterneuronal 3/9  epidural catheter × 2 neuronal 3/10 epidural catheter × 2neuronal 3/11 iv neuronal 3/20 im neuronal 3/22 im neuronal 3/23 im × 2neuronal 3/24 im non-neuronal 3/27 im non-neuronal 4/5  intrathecalneuronal 4/12 im neuronal 5/1  im neuronal epidural catheter × 4 5/2  imneuronal epidural catheter 5/10 epidural catheter × 4 neuronal 5/11epidural catheter × 4 neuronal 5/12 epidural catheter × 4 neuronal

Case Study 8

ReRo 1.3.4./007/221005/β, a twenty six year old subject, paraplegic as aresult of SCI at D6, was unable to move both lower limbs, although ableto sit without support. The subject had no bladder or bowel control, andhad sensation only from the inter mammary area upwards.

The subject was administered a pharmaceutical composition comprising hEScells and their derivatives according to the practice of the presentinvention about ten months after the injury. The subject regainedsensation in the lateral side of the body and up to the unbilious regionbilaterally from the axillary area to the hipbone. The subject can walkwith the aid of a walking frame and a caliper with motor power in thelegs.

The schedule of injections for this patient is shown in Table 39.

TABLE 39 Route of Date administration Cell types 9/26 test dose imneuronal 9/27 im neuronal and non-neuronal mixture 9/28 im neuronal andnon-neuronal mixture 9/29 im neuronal 9/30 im neuronal and non-neuronalmixture 10/1  im neuronal and non-neuronal mixture 10/3  im neuronal andnon-neuronal mixture 10/4  im non-neuronal intrathecal 10/5  m neuronal10/6  epidural neuronal 10/8  m non-neuronal 10/10  im neuronal 10/12 im neuronal deep spinal 10/15  iv neuronal 10/19  im non-neuronal 10/24 im neuronal and non-neuronal mixture 10/25  epidural neuronal 12/8  deepspinal non-neuronal 12/10  intrathecal non-neuronal 12/11  imnon-neuronal 12/12  deep spinal non-neuronal 12/13  im neuronal 12/14 epidural neuronal 12/15  im neuronal 2/13 im neuronal 2/15 epiduralneuronal 2/16 iv neuronal 2/17 im neuronal 5/24 im neuronal iv 5/25 imnon-neuronal iv neuronal 5/26 im neuronal iv × 2 non-neuronal, neuronal5/27 im × 2 non-neuronal iv × 2 neuronal 5/28 im neuronal, non-neuronal5/29 im neuronal, non-neuronal 5/30 epidural infusion neuronal 5/31 imneuronal

Case Study 9

ReRo 1.3.4./008/151005/β, a twenty seven year old subject suffered withtraumatic quadriplegia and had great difficulty talking and breathing,with rigid neck. The legs of the subject were paralysed and the subjecthad no finger movement and no sensation in the rest of the body.

The treatment via administration of a pharmaceutical compositioncomprising hES cells and their derivatives according to the practice ofthe present invention was started about two years after the injury. Thesubject experienced an improvement in neck movement, ease in speech, andimprovement in control of voice tone. The subject found breathing to beless cumbersome and motor function returned with some finger movement.The legs became less spastic and the subject was able to sit unaided.Toe movements also resumed and the subject can move the shoulders.

The schedule of injections for this patient is shown in Table 40.

TABLE 40 Route of Date administration Cell types 5/26 test dosenon-neuronal 5/27 im neuronal 5/28 im neuronal 5/29 im neuronal 5/30 imneuronal 5/31 im neuronal 6/1  im neuronal 6/8  im × 2 neuronalnon-neuronal 6/9  im neuronal 6/10 im × 2 neuronal non-neuronal 6/11 im× 2 neuronal non-neuronal 6/12 im × 2 neuronal non-neuronal 6/13 im × 2neuronal non-neuronal 6/14 intrathecal neuronal 6/15 im × 2 neuronalnon-neuronal 6/16 im × 2 neuronal non-neuronal 6/17 im × 2 neuronalnon-neuronal 6/18 im non-neuronal 6/19 im × 2 neuronal non-neuronal 6/20im × 2 neuronal 6/21 im × 2 neuronal non-neuronal 6/22 im × 2 neuronal6/23 iv × 2 non-neuronal 6/24 iv × 2 non-neuronal 6/25 iv × 2non-neuronal 6/26 epidural catheter × 2 neuronal 6/27 epidural catheter× 2 neuronal 6/28 epidural catheter × 3 neuronal 7/21 im neuronal ivnon-neuronal 7/22 im × 2 neuronal non-neuronal 7/23 im × 2 neuronal 8/22intrathecal neuronal

Case Study 10

ReRo 1.3.4./009/300106/α, a twenty five year old subject had a roadaccident and lost the ability to sit without support, the spine wasprone to buckling, the subject lost bladder and bowel control and thesubject suffered a total loss of power in the legs.

The subject was treated by administration of a pharmaceuticalcomposition comprising hES cells and their derivatives according to thepractice of the present invention about two years after the injury. Thesubject showed rapid improvement and can sit without support. Thesubject suffers no giddiness, has movement in both feet, regainedsensation in the finger tips and can experience chill flowing throughthe spine.

Case Study 11

ReRo 1.3.4./010/020206/β, a twenty six year old subject with a SCI atD12-L1 as a result of a road traffic accident was unable to stand withthe knees being contracted. The subject was able to sit without supportand had normal sensations in the limbs. The subject had no bladder orbowel control and had increased spasticity.

The subject was treated about twelve years after the injury byadministration of a pharmaceutical composition comprising hES cells andtheir derivatives according to the practice of the present invention.The subject experienced remarkable recovery in leg motor function withdecreased spasticity, exhibited a fully extendable right leg and areturn of strength to the left leg. The subject regained the ability tostand and walk with the aid of calipers and a walking frame. Bladder andbowel sensations also returned in the subject.

The schedule of injections for this patient is shown in Table 41.

TABLE 41 Route of Date administration Cell types 3/27 test dosenon-neuronal 3/29 im non-neuronal iv neuronal 3/30 im neuronal 3/31 imneuronal 4/1  im neuronal 4/2  im non-neuronal 4/3  im neuronal 4/4  imneuronal 4/5  caudal neuronal 4/7  epidural neuronal 4/8  im neuronal4/10 im neuronal 4/13 im neuronal 4/14 im neuronal 4/14 im neuronal 4/16im neuronal 4/18 iv non-neuronal 4/22 im neuronal 4/23 im neuronal 4/24epidural catheter neuronal 4/25 epidural catheter neuronal 4/26 ivneuronal 4/27 im neuronal 4/28 im neuronal 4/29 im neuronal 4/30 imneuronal 5/1  im neuronal 5/2  im non-neuronal 5/3  im neuronal 5/4  imneuronal 5/5  im neuronal 5/7  im neuronal 5/8  im non-neuronal 5/9  imnon-neuronal 5/10 im neuronal 5/11 caudal neuronal 5/12 im neuronal 5/13im neuronal 5/14 im neuronal 5/16 im non-neuronal 5/17 im neuronal 5/20epidural neuronal 5/21 im neuronal 5/22 im neuronal 5/23 im neuronal5/24 im neuronal

Case Study 12

The patient was a 26 year old male with paraplegia after an injurybetween D6-D8. He suffered a traumatic road accident in September 2004.He was totally bedridden with no feeling below the chest, no bladder orbowel control, and no sensation or motor power from chest downwards. Hehad a very deep bed sore in the lower back in which his sacrum could beseen.

The patient started hES cells on Apr. 27, 2006. Because of the bedsorehe could not undergo any O/T procedures and was given daily dosages ofcells intravenously and intramuscularly, and eventually had hES cellsapplied directly to his bedsore. He was also given intravenousinfusions.

On his 11^(th) day of treatment he could stand with full calipers (waistto ankle) and a walker and took 2 steps. As time continued his musclepower increased and after 4 months he could take up to 100 steps andcould stand for up to 20 mins. He is able to feel his legs and move themtoo. He is also able to feel bladder and bowel fullness and is able towalk now with just a knee brace and the walker for support. His bed sorehas healed and he has resumed his studies.

The schedule of injections for this patient is shown in Table 42.

TABLE 42 Route of Date administration Cell types 4/28 im neuronal (testdose) iv 4/29 iv neuronal im non neuronal 4/30 iv neuronal im nonneuronal 5/1  iv neuronal im non neuronal 5/2  iv neuronal im nonneuronal 5/3  iv infusion neuronal im non neuronal 5/4  iv infusionneuronal im non neuronal 5/5  iv neuronal im non neuronal 5/6  ivneuronal im non neuronal 5/8  iv infusion neuronal im 5/9  iv infusionneuronal im non neuronal 5/10 iv infusion neuronal im non neuronal 5/11iv neuronal im 5/12 iv neuronal im non neuronal 5/13 iv neuronal im 5/14iv neuronal im non neuronal 5/15 iv infusion non neuronal im 5/16 ivinfusion non neuronal im 5/17 iv neuronal im non neuronal 5/18 ivneuronal im non neuronal 5/19 iv neuronal im non neuronal 5/20 ivneuronal im non neuronal 5/21 iv neuronal im non neuronal 5/22 ivinfusion non neuronal im 5/23 iv infusion non neuronal im 5/24 ivneuronal im non neuronal 5/25 iv neuronal im non neuronal 5/26 ivneuronal im non neuronal 5/27 iv neuronal im non neuronal 5/28 ivneuronal im non neuronal 5/29 iv infusion neuronal im non neuronal 5/30iv infusion neuronal im non neuronal 5/31 iv neuronal im non neuronal6/1  iv neuronal im non neuronal 6/2  iv neuronal im non neuronal 6/3 iv neuronal im non neuronal 6/4  iv neuronal im non neuronal 6/5  ivinfusion neuronal im non neuronal 6/6  iv infusion neuronal im nonneuronal 6/7  iv neuronal im non neuronal 6/8  im neuronal iv nonneuronal 6/9  im neuronal iv non neuronal 6/10 im neuronal iv nonneuronal 6/11 im neuronal iv non neuronal 6/12 iv infusion neuronal 6/13iv infusion non neuronal im 6/14 iv neuronal im non neuronal 6/15 ivneuronal im non neuronal 6/16 iv neuronal im non neuronal 6/17 ivneuronal im non neuronal 6/18 iv neuronal im non neuronal 6/20 ivneuronal iv infusion im 6/21 iv neuronal non neuronal 6/22 iv nonneuronal (2) im 6/23 iv non neuronal (2) 6/24 iv non neuronal (2) 6/25iv non neuronal (2) 6/26 iv non neuronal iv infusion 6/27 iv nonneuronal iv infusion im 6/28 iv neuronal im 6/29 iv neuronal im nonneuronal × 2 6/30 iv neuronal im non neuronal × 2 7/1  iv neuronal imnon neuronal × 2 7/2  iv neuronal im non neuronal × 2 7/3  iv neuronaliv infusion non neuronal × 2 im 7/4  iv neuronal iv infusion nonneuronal im 7/5  iv neuronal × 2 im 7/6  iv neuronal × 2 im 7/7  ivneuronal im 7/8  iv neuronal im non neuronal × 2 7/9  iv neuronal im nonneuronal 7/11 iv neuronal iv infusion non neuronal im 7/12 iv neuronaliv infusion 7/17 im neuronal dressing non neuronal 7/18 iv infusion nonneuronal dressing 7/19 iv infusion non neuronal im 7/20 iv neuronal imnon neuronal dressing 7/21 iv neuronal im non neuronal dressing 7/22 ivneuronal im non neuronal dressing 7/23 iv neuronal im non neuronal 7/24iv neuronal im non neuronal 7/25 im neuronal dressing non neuronal × 27/26 m neuronal dressing non neuronal × 2 7/27 im neuronal non neuronal7/28 im non neuronal dressing 7/29 im neuronal dressing non neuronal7/30 im neuronal non neuronal 7/31 im non neuronal dressing 8/1  im nonneuronal × 2 dressing 8/2  im non neuronal × 2 dressing 8/3  im nonneuronal × 2 8/4  im non neuronal × 2 8/5  im non neuronal × 2 dressing8/6  im non neuronal × 2 8/7  im non neuronal × 2 dressing 8/8  im nonneuronal × 2 8/9  im non neuronal × 2 dressing 8/10 im non neuronal × 28/11 im non neuronal × 2 8/12 im non neuronal × 2 dressing 8/13 im nonneuronal × 2 8/14 im non neuronal × 2 dressing 8/15 im non neuronal × 2dressing 8/16 im non neuronal × 2 8/17 im non neuronal × 2 8/18 ivinfusion neuronal × 3 im 8/19 im neuronal × 3 dressing 12/5  im nonneuronal 12/6  iv infusion non neuronal 12/7  iv infusion non neuronalim dressing 12/8  epidural (intrathecal) neuronal 12/9  im non neuronal× 2 dressing 12/10  im non neuronal × 2 dressing 12/11  im non neuronal× 2 dressing 12/12  epidural (catheter) neuronal × 2 12/13  epidural(catheter) neuronal × 2 12/14  epidural (catheter) neuronal × 2 12/15 iv infusion neuronal, non neuronal im 12/16  iv infusion neuronal, nonneuronal

Case Study 13

The patient is a 22 year old male who suffered a horse riding accidentin June, 2006 and is a paraplegic at T-12, L-1. He had no power belowthe waist, no bowel or bladder control, and no sensation below waist.

The patient started treatment in September 2006 admitted for 6 weeks. Heresponded to hES cells and within a week he was able to stand and walk afew steps with full calipers (waist to ankle) and walker. He hasprogressed and is on the knee brace with which he can stand for up to 30mins with a walker. He now has full sensation and bowel and bladdercontrol. He also has good balance with the stick and also the crutches.He is able to go up and down the stairs with the caliper and thecrutches.

The schedule of injections for this patient is shown in Table 43.

TABLE 43 Route of Date administration Cell types 10/4  im non neuronal(test dose) 10/5  im non neuronal 10/6  im non neuronal 10/7  im nonneuronal 10/8  im non neuronal 10/9  iv infusion neuronal 10/10 ivinfusion neuronal 10/11 im non neuronal 10/12 im non neuronal 10/13 imnon neuronal 10/14 im non neuronal 10/15 im non neuronal 10/16 caudalneuronal 10/17 im non neuronal 10/18 im non neuronal 10/19 iv infusionnon neuronal im 10/20 iv infusion im non neuronal 10/21 im non neuronal10/22 im non neuronal 10/23 lumbar neuronal 10/27 im non neuronal 10/25im non neuronal 10/26 im non neuronal 10/27 im non neuronal 10/28 im nonneuronal 10/29 im non neuronal 10/30 epidural catheter neuronal 10/31epidural catheter neuronal 11/1  epidural catheter neuronal 11/2  im nonneuronal 11/3  im non neuronal 11/4  im non neuronal 11/5  im nonneuronal 11/6  im non neuronal 11/7  im non neuronal 11/8  im nonneuronal 11/9  caudal neuronal 11/10 im non neuronal 11/11 im nonneuronal 11/12 im non neuronal 11/13 iv infusion mixed im non neuronal11/14 im non neuronal iv infusion 11/15 im non neuronal 12/31 iv mixed 1/10 im non neuronal  1/11 iv infusion mixed  1/12 iv infusion mixed 1/13 im non neuronal  1/14 im non neuronal  1/15 caudal neuronal  1/16im non neuronal  1/17 im non neuronal  1/18 im non neuronal  1/19 lumbarneuronal  1/20 im non neuronal mixed  1/21 im mixed  1/22 im mixed  1/23epidural catheter neuronal  1/24 im neuronal iv  1/25 im neuronal  1/26infusion mixed im neuronal  1/27 infusion mixed im neuronal

Case Study 14

The patient is a 26 year old female traumatic paraplegic at T-12, L-1who suffered a road traffic accident in September 2004. She had nofeeling below the chest and she had no bowel or bladder control. Therewas no motor power in either leg and she was wheel chair bound.

The patient started hES cells in March 2006. She was able to stand andwalk a few steps with full calipers (waist to ankle) and walker after 9days and continued to progress. Her current status is that she can walkcontinuously with caliper and walker and also stand using the kneebrace. Bowel and bladder sensations have been restored with control andwithout the usage of any catheterization or suppository. Sensation hasimproved to the ankle levels.

The schedule of injections for this patient is shown in Table 44.

TABLE 44 Route of Date administration Cell types 3/27 im non neuronal(test dose) 3/29 iv neuronal im non neuronal 3/30 im neuronal 3/31 imneuronal 4/1  im neuronal 4/3  im neuronal 4/4  im neuronal 4/5 Epidural (caudal) neuronal × 2 4/7  Epidural (intrathecal) neuronal × 24/9  im neuronal 4/10 im neuronal 4/12 im neuronal 4/13 im neuronal 4/14im neuronal 4/15 im neuronal 4/16 im neuronal 4/17 im neuronal 4/18 ivnon neuronal 4/20 im neuronal 4/21 im neuronal 4/22 im neuronal 4/24Epidural (catheter) neuronal × 4 4/25 Epidural (catheter) neuronal × 44/26 iv (catheter) neuronal × 4 4/27 im neuronal 4/28 im neuronal 4/29im neuronal 4/30 im neuronal 5/1  im neuronal 5/2  im non neuronal 5/3 im neuronal 5/4  im neuronal 5/5  im neuronal 5/7  im neuronal 5/8  imnon neuronal 5/10 im neuronal 5/11 EPI (caudal) neuronal 5/13 IMneuronal 5/14 IM neuronal 5/16 IM non neuronal 5/17 IM neuronal 5/18 EPI(catheter) neuronal × 2 5/19 EPI (catheter) neuronal × 2 5/20 EPI(catheter) neuronal × 2 5/21 im neuronal 5/22 im neuronal 5/23 imneuronal 5/24 im neuronal 5/25 im neuronal 10/25  im non neuronal mixed10/26  im non neuronal iv infusion mixed 10/27  im non neuronal 10/28 im non neuronal 10/29  im non neuronal 10/30  caudal neuronal 10/31  imnon neuronal 11/1  im non neuronal × 2 11/2  im non neuronal × 2 11/3 lumbar neuronal 11/4  infusion neuronal 11/5  im non neuronal × 2 11/6 im non neuronal × 2 11/7  im non neuronal × 2 11/8  epidural catheterneuronal 11/9  epidural catheter neuronal 11/10  epidural catheterneuronal 11/11  im non neuronal × 2 11/12  im non neuronal infusionmixed 11/13  caudal neuronal infusion mixed 11/14  im neuronal

Human Embryonic Stem Cell Culture Methodology

The hES cells used as the starting material for the cell lines developedunder the present invention are derived from a 2 to 7 day old embryoprior to its implantation into the uterus, e.g., a 2 to 4 day oldembryo, e.g., a 3 day old embryo.

For embryo isolation, the ova are collected with consent from humandonors who are undergoing a regular IVF cycle. The ova are fertilized bythe sperm and cultured by conventional methods to obtain the embryos.Extra embryos are incubated for variable periods for development of thecell line.

The embryo is suspended in a small amount of minimal essential medium(e.g., RPMI, e.g., RPMI 1640 with 2.2 g/L sodium bicarbonate) and thehES cells are isolated from the embryo by mechanical means (e.g.,shaking) Additional medium is added to the isolated cells along with aprogestin and a βhCG agonist. In one embodiment, progesterone (16-64 μlof 250 mg/ml) and βhCG (16-64 μl of 5000 iu/ml) are added. The isolatedcells are cultured for 12-48 hours, e.g., 24 hours, at a temperature ofbetween 34-38° C., in an environment of 3.5-6% carbon dioxide.

In one embodiment, the hES cells are cultured under anaerobic orsubstantially anaerobic conditions in order to expand the cells whilepreventing differentiation. “Substantially anaerobic” is defined as lessthan about 10% O₂, e.g., less than 8%, 6%, 4%, 2%, or 1% O₂. Low oxygenconditions may be created using multi-gas (CO₂, O₂, N₂) cell incubatorsin which the oxygen level may be set between 0% and 20% by replacing theambient air with nitrogen gas. Examples of multi-gas incubators includethe Fisher 11-730 series, the Napco 7000 series, the Sanyo MCO series,the Jouan IG750, and the Heraeus Heracell 150. In another embodiment,the cells are cultured in a CO₂ incubator and the level of oxygen iscontrolled by the shape and position of the flask in which the cells arecontained. For example, when a cell culture flask is maintained in avertical position, much of the culture medium in the flask issubstantially anaerobic. In contrast, when the cell culture flask ismaintained in a horizontal position, the culture medium is substantiallyaerobic. The level of oxygen in the culture medium can be varied betweensubstantially anaerobic and substantially aerobic by altering the shapeand/or position of the cell culture flask within the incubator or theamount of medium in the flask (e.g., the amount of head space).

During the expansion stage, flasks may be maintained in a verticalposition rather than horizontal. In one embodiment, incubation iscarried out in a culture vessel where the volume is almost completelyoccupied by the medium and the vessel is kept in a vertical position. Asa consequence, a substantial proportion of the cells do not adhere tothe walls of the culture flask, and the culture medium is substantiallyanaerobic.

Under such growth conditions, cell-doubling or replication cycles are attheir maximum, and cellular differentiation processes are substantiallyinhibited. Cell replication cycles during expansion proceed for 12-48hours, e.g., 24 hours.

For passaging or re-culture of the hES cells, the cell suspension iscentrifuged, the cell pellet is resuspended in a small amount of freshculture medium comprising progesterone and βhCG, the cells are aliquotedand additional fresh medium without progesterone and βhCG is added. Inone embodiment, the ratio of the aliquot of incubated stem cells to thefresh cell medium is about 1:3.5 to about 1:35 volume/volume. The cellsare re-incubated at 34-38° C. in a water jacketed incubator supplementedwith an atmosphere of 3.5-6% carbon dioxide under substantiallyanaerobic conditions for 12 to 48 hours, e.g., 24 hours. At this pointthe cells may be passaged for continued expansion or stored for futureuse.

hES cells cultured under such conditions remain in a largelyundifferentiated form as determined by alkaline phosphatase activity andthe absence of any markers characteristic of cellular specialization ordifferentiation. However, it may be noted that no cell population isobtained which is completely differentiated or completelyundifferentiated. The cell population is a mix of differentiated andundifferentiated cell lines, with a ratio ranging from about 4:1 toabout 10:1 undifferentiated to differentiated cells.

For storage of the expanded hES cells, e.g., in a deep freezer or inliquid nitrogen, a cryopreservation agent, including but not limited to0.2-2% (w/v) dimethyl sulphoxide (DMSO), may be added to the culturemedium. The level of DMSO used is less than typically used forcryopreservation (e.g., 2-84 μl/0.5 ml tube) to avoid inducingdifferentiation or damage to the cells. The ratio of the amount ofcryopreservation agent to that of culture medium containing stem cellsmay vary from about 1:500 to about 16:1000. Other cryopreservationagents include without limitation glycerol, propanediol, butanediol,ethanol, glucose, D-glucose, sucrose, trehalose, mannitol, paparavine,formamide, probuchol, curcumin, polyvinylpyrrolidone, polyethyleneglycol, chondroitin sulfate, glycosaminoglycan dimethyl sulfoxide,glutamine, and sodium pyruvate. The freezing temperature for the cellsuspension may be varied from about −15 to about −80° C., e.g., about−15 to about −40° C. In contrast to other techniques for the storage ofhuman embryonic stem cells and their derivatives, there is no need forflash freezing or storage in “straws”. After storage, the cells may beprepared for continued expansion or for differentiation by collectingthe cells by centrifugation and resuspending the cell pellet in freshmedium.

Partial differentiation of the expanded hES cells (e.g., after expansionof freshly isolated hES cells or storage of previously expanded cells)is carried out by sedimenting the cell suspension in a centrifuge (e.g.,at between 700-1400 rpm for 5-12 minutes), removing and discarding thesupernatant, and resuspending the cells in a small amount of freshmedium (e.g., RPMI) containing a progestin and a βhCG agonist. The cellsare then aliquoted and additional culture medium (e.g., a minimalessential medium such as RPMI or DMEM, e.g., 1× glutamic acid free DMEMor DMEM with 4.5 g/L glucose and 3.7 g/L sodium bicarbonate) withoutprogestin or βhCG is added. The cells are cultured under substantiallyaerobic conditions (e.g., in a flask in a horizontal position) for 12 to48 hours, e.g., 24 hours. “Substantially aerobic” is defined as at leastabout 15% O₂, e.g., at least about 18% or 20% O₂. Under substantiallyaerobic conditions the hES cells will begin to differentiate. Thedifferentiation pathway for the hES cells is dependent on the culturemedium used during the differentiation stage. To produce neuronalprogenitor cells, the hES cells are cultured in DMEM or its equivalent.To produce progenitor cells other than neuronal progenitors, the hEScells are cultured in RPMI or its equivalent. After the 12-48 hourreplication cycle, the cells may be collected and resuspended in freshmedium to continue differentiation. In general, the differentiationstage should not last more than 48-72 hours as further differentiationbeyond this time produces cells that are not suitable fortransplantation using the methods of the present invention.

Once the cells are partially differentiated, the cells can either bestored for future use by adding a cryopreservative and storing the cellsat −15 to about −80° C. as described above or preparing the cells forusage in the methods of the invention. To prepare the cells for usage,the cells are aliquoted, fresh medium is added (e.g., DMEM or RPMI asappropriate) and the cells are cultured for 12-48 hours, e.g., 24 hours,under substantially anaerobic conditions to prevent furtherdifferentiation. The cells are then aliquoted into fresh medium forcontinued expansion or collected by centrifugation and resuspended in abiocompatible solution (e.g., saline) in preparation fortransplantation. At this point the cells may be transplanted intopatients or stored at +4° C. to −80° C. for future transplantation.Storage may be in any suitable container including but not limited totest tubes, vials, syringes, etc. that may facilitate transportation orclinical use. In one embodiment, the resuspended cells in biocompatiblesolution are stored in a ready-to-use form (e.g., in a prefilledsyringe). When needed, aliquots of cells are thawed naturally withoutthe need for water baths or incubators.

Under the described storage conditions in a biocompatible solution, cellviability of >40% is observed after re-thawing even after six months ofstorage, with no detectable genotypic (genetic instability) orphenotypic alterations such as aneuploidy or heteroploidy.

At each stage of expansion, differentiation, and storage, an aliquot ofthe cells is tested for viability via trypan blue exclusion andmicroscopic examination. An aliquot of the cell culture is also testedfor microbiological contamination. A further aliquot of the cellsuspension is also placed on a haemocytometer and examinedmicroscopically in order to determine the cell density. A furtheraliquot is also taken for testing alkaline phosphatase activity as amarker for the state of differentiation of the cell culture.

During storage, the cells may be tested once a month for theirkaryotype, to test for genetic instability arising as a result of theculture methodology.

EXAMPLES Example 1

For embryo isolation, ova were collected with consent from a human donorwho underwent a regular IVF cycle, which produced 8 follicles. The ovawere fertilized by the sperm and cultured by conventional methods toobtain the embryos. Three of the embryos were transplanted into thedonor. The extra embryos were incubated for variable periods fordevelopment of an hES cell line.

The embryo, either intact or in broken condition, was suspended inculture media. Further, 84 μl of progesterone (250 mg/ml) and 84 μl ofβhCG (5000 iu/ml) were added. The media with the embryonic cells weretested for any contamination, and any infected embryo was discarded. Thecells of the embryos in this form were used for expansion and storage.

After one day of incubation, 1 ml of the embryonic cell containing mediawere introduced in a 40 ml cell culture medium (DMEM or RPMI) in a 50 mlcontainer along with progesterone and βhCG. The culture medium alongwith the stem cells was incubated in a horizontal position at ambienttemperature in an environment containing carbon dioxide. Table 45 showssome of the experimental conditions followed for this method.

TABLE 45 (a) (k) Embryo (b) (c) (d) (e) (f) (g) (h) (i) (j) Incubation(l) S. stage in Embryo Cell Amount Addition Incubation aliquot CellAmount Container temp % CO2 in No. days state Media of (c) to Media timeof (f) Media of (h) volume degree C. environment 1 2 Intact IVF media2.5 ml FF 24 hrs — — — — 36 4 2 2 Intact IVF media 2.5 ml A 24 hrs — — —— 36.5 4.5 3 3 Broken RPMI 2.5 ml A 24 hrs 0.5 ml RPMI 45 ml 50 ml 37 54 3 Broken RPMI 2.5 ml A& B 24 hrs 1.0 ml RPMI 46 ml 50 ml 38 5 5 3Broken DMEM 2.5 ml B 24 hrs 0.5 ml DMEM 35 ml 50 ml 37 5

TABLE 46 (a) Amount (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) S. ofESC Cell Amount Addition Container Incubation % CO2 in IncubationAliquot Culture Container Storage No. culture Media of (b) to Mediavolume temperature environment time of (h) medium volume temp 1 0.5 mlRPMI 35 ml A 50 ml 36.5 5 24 0.5 ml RPMI 0.5 ml   −72 2 1.0 ml RPMI 38ml B 50 ml 37 5 24 .25 ml RPMI 0.5 ml −−20 3 1.5 ml DMEM 45 ml AB 50 ml37 5 48 1.0 ml DMEM 1.5 ml −−20 4 2.0 ml RPMI 46 ml A 50 ml 37.2 5 481.5 ml RPMI 1.5 ml −−18 5 2.5 ml DMEM 45 ml A 50 ml 36.8 5 24 0.5 mlDMEM 1.5 ml −−72 FF = follicular fluid aspirated along with the ovumduring the IVF cycle, 16-84 μl; A = progesterone; B = βhCG

After 24 hrs of incubation, equal numbers of nucleated (partlydifferentiated) cells and blank (undifferentiated) cells were observed.Aliquots of about 0.5 ml were taken for storage at this stage. After 48hrs of incubation, oblong shape cells with few strands were observedwith DMEM media. However, when RPMI media was used, many nucleated cellswith varied shapes were observed. The volume of the container was foundto be filled up to the 37 ml mark after the 48 hr incubation. Aliquotsof 0.5 ml were taken for storage at this stage. After 72 hrs ofincubation in DMEM, long strands of cells cross linked like nerve tissuewere observed. The RPMI media incubation produced a few small sizedcells and a few cells accumulating around a central cell. Aliquots of0.5 ml were taken for storage at this stage.

An aliquot of the embryonic cells grown in culture containing media wereintroduced into more cell medium (RPMI or DMEM) with other testcomponents, in a sterile container (Tarsons steriflask). The stem cellswere incubated at ambient temperature in an environment of carbondioxide in horizontal position for various time periods ranging from 18hrs to 5 days. After 24 hrs of incubation, aliquots of about 0.5 ml wereagain introduced into more culture media and reincubated. Aliquots weretaken for preparing ready to use compositions as well as for storageafter different incubation stages. Table 46 depicts some experimentalconditions followed for expansion of hES cells.

Example 2

The embryo, either intact or in broken condition, was suspended inculture media. Further, 84 μl of progesterone and 84 μl of βhCG wereadded. The media with the embryonic cells were tested for anycontamination, and any infected embryo was discarded. The cells of theembryos in this form were used for expansion and storage.

After one day of incubation, 1 ml of the embryonic cell containing mediawas introduced in a 46 ml cell culture medium (DMEM or RPMI) in a 50 mlcontainer along with progesterone and βhCG. The stem cells wereincubated in a vertical position at ambient temperature in anenvironment of carbon dioxide. Table 47 shows some of the workingexample variables used for this experiment.

TABLE 47 (A) Embryo (B) (C) (D) (E) (F) (G) (H) (I) (J) (K) (L) S. stagein Embryo Cell Amount Addition to Incubation aliquot Cell AmountContainer Incubation % CO2 in No. days state Media of (c) Media time of(f) Media of (h) volume temp environment 1 2 Intact IVF media 2.5 ml FF24 hrs — — — — 36 4 2 2 Intact IVF media 2.5 ml A 24 hrs — — — — 36.54.5 3 3 Broken RPMI 2.5 ml A 24 hrs 0.5 ml RPMI 45 ml 50 ml 37 5 4 3Broken RPMI 2.5 ml B 24 hrs 1.0 ml RPMI 46 ml 50 ml 38 5 5 3 Broken DMEM2.5 ml A& B 24 hrs 0.5 ml DMEM 35 ml 50 ml 37 5

TABLE 48 (a) Amount (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) S. ofESC Cell Amount Addition Container Incubation % CO2 in IncubationAliquot Culture Container Storage No. culture Media of (b)) to Mediavolume temperature environment time hrs of (h) medium volume temp 1 0.5ml RPMI 35 ml A 50 ml 36.5 5 24 0.5 ml RPMI 0.5 ml −−40 2 1.0 ml RPMI 38ml A 50 ml 37 5 24 .25 ml RPMI 0.5 ml −−20 3 1.5 ml DMEM 45 ml B 50 ml37 5 48 1.0 ml DMEM 1.5 ml −−18 4 2.0 ml RPMI 46 ml A 50 ml 37.2 5 481.5 ml RPMI 1.5 ml −−20 5 2.5 ml DMEM 45 ml AB 50 ml 36.8 5 24 0.5 mlDMEM 1.5 ml −−72

After 24 hrs of incubation, nucleated cells (partly differentiated) andblank cells (undifferentiated) were observed in a ratio of about 1:4.After 48 hrs of incubation, nucleated cells and blank cells in the ratio1:2 were observed. Aliquots of 0.5 ml were taken for storage at the 24hr and 48 hr stage.

0.5 ml of the embryonic cells grown in culture containing media wereintroduced in 46 ml cell medium (RPMI or DMEM) with other testcomponents, in a 50 ml sterile container (Tarsons steriflask). The stemcells were incubated in a horizontal position at ambient temperature inan environment containing carbon dioxide. After 24 hrs of incubation,aliquots of 0.5 ml were again introduced into 46 ml of culture media andreincubated. Table 48 depicts some experimental conditions followed forexpansion of hES cells.

Aliquots were taken for preparing ready to use compositions as well asfor storage after the 24 hr incubation stage.

Example 3 Storage

For storage, hES cells were taken in a 0.5 ml storage tube and acryopreservation agent such as 0.2% DMSO in a quantity of 16 μl wasadded and after gentle shaking were stored at −20° C. The cells werethawed naturally for making ready to use compositions or for furtherexpansion. The viability of the thawed cells was tested and 64% to 84%of the cells were found to be viable. The stored cells were tested forcontamination as well as viability after regular intervals. Table 49shows the actual parameters followed for storage in five differentexperiments as well as the viability of the cells after thawing.

TABLE 49 Amount of Cell DMSO Viability of S. No. suspension quantityDMSO % Media used thawed cells 1 0.5 ml 16 μl 0.2 NaCl 74% 2 0.5 ml 32μl 1.4 NaCl 66% 3 0.5 ml 64 μl 0.4 NaCl 70% 4 0.5 ml 48 μl 0.2 NaCl 78%5 0.5 ml 16 μl 0.2 NaCl 88%

Example 4

Various tests were carried out for checking any contamination orinfection or abnormality in the embryonic cells at various intervals ofthe expansion and storage stages and to identify the hES derivativesthat were present in each culture. The different tests include those forHIV, HbSAg (for hepatitis), conventional PCR for Kochs test (fortuberculosis), chromosomal analysis by CG method of giemsa banding,Bilirubin by the Jendrasiik and Grof method and Albumin using BCG Dyebinding method (for liver progenitor cells), insulin via CLIA method(for pancreatic progenitor cells), neurofilament by immunohistochemicalmethod (for neuronal progenitor cells), CD34 testing byimmunohistochemical method (for hematopoietic progenitor cells),Alkaline Phosphatase via PNPP method (for undifferentiated cells),Histopathology tests (for cell identification by morphology), etc. Theculture condition was tested by the manual culture plate method andmanual sensitivity and identification with versatrek/AP1 was carried outfor fungal infection. All the tests were carried out and the cell countand viability of the cells were checked at each stage. Table 50 providesthe results of some of the tests.

Example 5 Preparation of hES Cells for Transplantation

15 ml of stem cell suspension was taken and centrifuged at 1000 r.p.m.for 7 minutes. The supernatant was discarded. 2 to 15 ml of saline wasadded to the pellet and the stem cells thus suspended. The suspensionwas checked for microbial contamination. A viability test was also done.

Example 6 Storage of hES Cells in Ready-to-Use Form

Containers (syringe, test tube, flask, vial) filled with thetransplantable hES cell suspension were labeled and stored at −20° C.The cold chain was maintained up to the transplantation stage. Thesuspension was thawed naturally immediately before transplantation.

TABLE 50 Batch Alk S Cell Count Viability Culture Code Bilirubin HIVHbSAg Phos PCR Albumin insulin Neurofil Histo (in millions) % State B77— −ve −ve — −ve — — — — 3.86 72 Sterile B78 — −ve −ve — −ve — — — — 3.1870 Sterile B79 — −ve −ve — −ve — — — — 3.92 70 Sterile B80 — −ve −ve —−ve — — — — 3.96 68 Sterile L56 — −ve −ve — −ve — — — — 3.12 72 SterileL57 — −ve −ve — −ve — — — — 3.84 72 Sterile B81 — −ve −ve — −ve — — — —3.16 70 Sterile B82 — −ve −ve — −ve — — — — 3.72 68 Sterile B83 — −ve−ve — −ve — — — — 3.68 74 Sterile B84 — −ve −ve — −ve — — — — 3.72 72Sterile B85 — −ve −ve — −ve — — — — 3.86 76 Sterile B86 — −ve −ve — −ve— — — — 3.92 78 Sterile L58 — −ve −ve — −ve — — — — 3.86 82 Sterile L591.27 −ve −ve 97 −ve 1.9 13.2 — — 3.12 70 Sterile B87 — −ve −ve — −ve — —— — 3.16 72 Sterile B88 — −ve −ve — −ve — — — — 3.84 70 Sterile L1/V 1 —−ve −ve — −ve — — — — 3.68 78 Sterile L1/V 2 — −ve −ve — −ve — — — —3.72 74 Sterile B89 — −ve −ve — −ve — — — — 3.68 72 Sterile B90 0.05 −ve−ve 14 −ve 0.15 — — — 3.76 70 Sterile B91 — −ve −ve — −ve — — — — 3.6468 Sterile B92 — −ve −ve — −ve — — — — 3.72 72 Sterile B93 — −ve −ve —−ve — — — — 3.74 70 Sterile B94 — −ve −ve — −ve — — — — 3.92 72 SterileB95 — −ve −ve — −ve — — — — 3.86 70 Sterile −ve = negative test result —= not done

Cell Lines

Subcultures were repeated more than 100 times to establish hES cells ofthe present invention. Further, the subcultures were tested for anycontamination/infection at each stage of further expansion or storage.The hES cell lines were found to be stable and without any abnormalityfor over five years of sub culturing cycles.

The present invention is not to be limited in scope by the specificembodiments and examples, which are intended as illustrations of anumber of aspects of the invention and any embodiments which arefunctionally equivalent are within the scope of this invention. Thoseskilled in the art will know, or be able to ascertain using no more thanroutine experimentation, many equivalents to the specific embodiments ofthe invention described herein. These and all other equivalents areintended to be encompassed by the following claims. All patents, patentapplications and publications cited herein are fully incorporated byreference.

1.-42. (canceled)
 43. A method of partially differentiating hES cells,derivatives of hES cells, or combinations thereof, free of feeder cells,animal products or growth factors other than a βhCG agonist or aprogestin, leukaemia inhibitory factor, supplementary mineralcombinations, amino acid supplements, vitamin supplements, fibroblastgrowth factor, membrane associated steel factor, soluble steel factor,and conditioned media, comprising: (a) introducing hES cells,derivatives of hES cells, or combinations thereof in a cell culturemedium consisting of minimal essential medium; and (b) incubating thecells at a temperature of about 34° C. to about 38° C. in an environmentof about 3.5% to about 6% carbon dioxide for about 12 hours to about 48hours.
 44. The method of claim 43, wherein said cell culture medium isRPMI or DMEM.
 45. The method of claim 43, wherein said incubation iscarried out in a water jacketed cell culture incubator.
 46. The methodof claim 43, wherein said cells in culture medium are incubated in abiocompatible container under substantially aerobic conditions.
 47. Themethod of claim 46, wherein said cells differentiate on proliferation.48. The method of claim 43, further comprising testing the partiallydifferentiated cells for any contamination.
 49. The method of claim 43,wherein said culture process is carried out in biocompatible containers.50. The method of claim 43, wherein the ratio of cells to the cellmedium is about 1:3.5 to about 1:35.
 51. The method of claim 43, whereinsaid incubation is carried out in a substantially aerobic environment.52. The method of claim 51, wherein said incubation is carried out in abiocompatible container and the container is kept in a horizontalposition. 53.-117. (canceled)